RU2780179C2 - Mesenchymal stem cells originating from perinatal tissue: method for their production and use - Google Patents

Abstract

FIELD: cell biology; medicine.

SUBSTANCE: present invention relates to the field of cell biology and medicine, in particular to the production of CD106^high CD151⁺ Nestin⁺ mesenchymal stem cells (hereinafter – MSC) originating from placental and embryonic tissues and increase in a level of expression of CD106 non-differentiated MSC, as well as cultures of such MSC cells, pharmaceutical and cosmetical compositions containing them. For the production of the specified MSC and increase in the expression of CD106, non-differentiated MSC are cultivated in a cultural medium containing a mixture of Ilβ and IL4. The resulting cell cultures and compositions are used for the treatment of diabetes, aplastic anemia, decompensated liver cirrhosis, for angiogenesis induction, as well as for regeneration of skin and mucosa cells, improvement of skin and mucosa appearance, and treatment of skin damage or disorder. The cell culture, according to the invention, is used in administration systems, which are micro- and nanovesicles of biopolymers, lipids, or nanoparticles.

EFFECT: present invention allows for an increase in the yield of functional MSC obtained from all placental and extraembryonic tissues, due to a more efficient cultivation system.

28 cl, 8 dwg, 8 tbl, 9 ex

Description

PRIOR ART

Mesenchymal stem cells (MSCs) are known to be useful in regenerative medicine and tissue engineering. In adults, bone marrow, adipose tissue, dental pulp, and menstrual blood are the main sources of MSC. They can also be obtained by culturing placental or umbilical cord cells for 3-4 weeks in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with FCS (fetal calf serum) ([12], [13], [14]).

MSCs have the potential for mesodermal, ectodermal, and endodermal differentiation. MSCs also have immunosuppressive properties. MSCs inhibit or stop the maturation of dendritic cells and the proliferation of T cells, B cells, and NK (natural killer) cells. Their immunomodulatory action is mediated by the cytokines and chemokines they secrete. Several groups have demonstrated that MSCs within placental tissue exhibit developmental plasticity towards many lineages in vitro and in vivo . MSCs derived from placental tissue (including the umbilical cord, cord blood, placenta, chorion, amnion, and amniotic fluid) show positive expression of CD29, CD44, CD73, CD90, and CD105, negative expression for hematopoietic surface markers CD11b, CD19, CD34, and CD45 , and negative expression for the endothelial surface marker CD31. In addition, placental tissue-derived MSCs, under suitable conditions (eg, complete medium), are capable of transdifferentiating into cells of all three germ layers.

It has also been shown that MSCs derived from perinatal tissue have broad immunoregulatory abilities and are able to influence both adaptive and innate immune responses. These MSCs inhibit immune cell proliferation and maturation and suppress immune responses both in vitro and in vivo in a manner not limited by MHC (major histocompatibility complex). Therefore, these MSCs are considered to be hypoimmunogenic, showing low levels of HLA (human leukocyte antigen) class I expression, no expression of HLA class II, and no expression of co-stimulatory molecules including CD40, CD80 and CD86. Basically, these MSCs could have widespread immunomodulatory effects on cells of both the innate and adaptive immune systems. Ex vivo expanded placental MSCs have also been shown to suppress the activity of a wide range of immune cells including T cells, natural killer T cells (NKT), dendritic cells (DC), B cells, neutrophils, monocytes, macrophages, and etc.

MSCs derived from human perinatal tissue are also safe according to numerous clinical trial reports. The safety and baseline efficacy of umbilical cord-derived MSC (UC-MSC) transfusions were evaluated in patients with exacerbation of chronic liver failure (ACLF) associated with hepatitis B virus (HBV) infection. These results indicated that MSC transfusions are clinically safe and may serve as a novel therapeutic approach for patients with HBV-associated ACLF [1]. The safety and efficacy of human UC-MSCs in the treatment of rheumatoid arthritis (RA) was also evaluated. These results indicated that no serious adverse effects were observed during or after the infusion. In addition, MSC treatment induced significant disease remission as measured by the Joint Disease Activity Index 28 [2]. The researchers evaluated the safety and feasibility of intramyocardial injection of MSC in nine patients shortly after acute myocardial infarction (AMI) during short-term and 5-year follow-up [3]. These results indicated that intramyocardial injection of MSC in patients shortly after AMI is possible and safe up to 5 years of follow-up. A prospective, double-blind, randomized, placebo-controlled, multicenter study to determine the safety of MSCs in patients with critical limb ischemia showed that MSCs are also safe when injected intramuscularly at a dose of 2 million cells/kg body weight [4]. In any of the MSC implantation sites, complications in the form of neoplasms were not detected. Hongye Fan et al ([15]) showed that transplantation of IL1β (interleukin-1beta) primed MSCs has an increased therapeutic efficacy in DSS-induced murine colitis, which depends on their increased immunosuppressive abilities and increased migratory ability. WO 2014/093948 also discloses the therapeutic value of purified MSCs.

However, the definition of MSCs has always been controversial, as there is no specific or unique cell surface marker that uniquely identifies them. Until now, MSCs have been defined using a combination of phenotypic cell surface protein markers, plastic, attached, fibroblast-like growth, and functional properties. However, according to these cell surface markers, there are several different subpopulations, and these different subpopulations show different biological characteristics, biological functions, and not all are effective in treatment protocols. For example, some populations of MSCs are stimulated by IL1β ([12]), while other populations are not stimulated (CD106-positive MSCs in [15]). In addition, the proliferation of some MSC populations is inhibited by IL4 ([12]), while others are induced to proliferate in the presence of this cytokine ([13]). Therefore, there is an urgent need to identify a population of functional MSCs that have clinical efficacy and to standardize their method of preparation. It is also important to establish a protocol that can be used interchangeably on each placental and extra-embryonic tissue. More specifically, it is important to identify a method of production that produces therapeutically effective MSC populations from either placental tissue or umbilical cord fragments.

In the meantime, there is now a great need for human MSCs for various therapeutic applications, but insufficient market availability. Therefore, there is a need for an industrial scale method that gives a high yield of functional MSCs. There is also a need for an efficient culture system that gives optimal yield at an affordable cost, thereby reducing the gap between supply and demand for MSCs derived from all placental and extra-embryonic tissues.

The present application satisfies all these needs and more by providing an optimal method for obtaining and purifying MSC subpopulations of different origins (either from placental tissue or umbilical cord) and ultimately propagating said subpopulation using the lowest possible number of passages and the minimum number of population doublings. . This method results in the isolation of highly efficient MSCs from the placental tissue and from the umbilical cord tissue, which have the ability to differentiate towards different lineages. This method reproducibly produces a significant number of MSC cells that are suitable for clinical use.

DETAILED DESCRIPTION OF THE INVENTION

Example 1 (see below) discloses a production method for generating human placenta-derived CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) on an industrial scale, providing an MSC yield of at least 1×10 ⁵ cells/cm ² . which are obtained in a GMP (Good Manufacturing Practice) compliant facility and which are suitable for allogeneic use. These placental-derived CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) contain over 95% of cells that express positive markers CD73, CD90, CD105 and CD166 and less than 2% of cells that express negative markers CD45, CD34 and HLA-DR.

This method is useful for obtaining cells that can be used in transplantation trials in a human subject or in an animal.

It includes the following two general stages:

(i) culturing mesenchymal stem cells derived from a biological tissue or fluid in a first culture medium devoid of growth factors, so as to obtain a population of cultured undifferentiated mesenchymal stem cells,

and

(ii) contacting said population of cultured undifferentiated mesenchymal stem cells with a second culture medium containing pro-inflammatory growth factors or inflammatory mediators, thereby generating CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells useful for transplantation into a subject in need thereof.

In a first aspect, the invention relates to a method for in vitro production of CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) on an industrial scale, said method comprising culturing a population of undifferentiated MSCs in a culture medium containing pro-inflammatory growth factors or inflammatory mediators.

Said " undifferentiated MSC population " can be obtained by selecting mononuclear cells present in a biological tissue or fluid and growing them in a first culture medium. These mononuclear cells can be obtained by any conventional methods, for example, enzymatic cleavage or explant cultivation of pieces of perinatal tissue [10], or isolation from biological fluids [11].

Explant culture is a particularly preferred method for obtaining MSC from umbilical cords as described in Example 3 below.

Typically, this method requires the removal of the sample from the transport solution to cut it into pieces (roughly 2-3 cm in length) for disinfection with antibiotics and antifungal agents, which are later washed to isolate the epithelial membrane and distribute pieces of this membrane into vials for attachment ( preferably without medium, at room temperature), before carefully adding complete medium to attached explants and keeping them incubated at 37°C for several days. The migrating cells are eventually harvested using suitable instruments and maintained in culture in a suitable first medium (see below) until they reach the target confluence.

Said " first culture medium " may be any of the classical media commonly used to promote the growth of living primary cells. Preferably it does not contain any growth factors or any differentiation factors.

The person skilled in the art is well aware of what types of culture media can be used as "first culture media". They are, for example, DMEM (Dulbecco's Modified Eagle's Medium), DMEM/F12, MEM (Minimum Culture Medium), alpha-MEM (α-MEM), IMDM (Iskov's Modified Dulbecco's Medium), or RPMI. Preferably, said first culture medium is DMEM (Dulbecco's Modified Eagle's Medium) or DMEM/F12 (Dulbecco's Modified Eagle's Medium: F-12 Nutrient Formula).

More preferably, said first culture medium contains 2-20% or 2-10% fetal calf serum. Alternatively, said first culture medium may contain 1-5% platelet lysate. The most preferred medium contains 2-20% or 2-10% fetal calf serum and 1-5% platelet lysate.

It is also possible to use as the first culture medium a medium which is devoid of serum or platelet lysate, provided that it contains other suitable agents that favor the growth of living primary cells.

In a preferred embodiment, said " biological tissue " is any part of the placental tissue or umbilical cord. In particular, it may include or consist of placental villi, amniotic membrane or placental chorionic membrane. It can also represent the Wharton jelly located in the umbilical cord. It may include veins and/or arteries, or be devoid of them.

In another embodiment, said " body fluid " is a sample of cord blood, placental blood, or amniotic fluid that has been collected in a harmless manner from a woman or, in general, a mammal. For example, tissue and fluid data can be obtained after the birth of a child or offspring, without any invasive procedures.

Said population of undifferentiated MSCs is preferably a population of mesenchymal stem cells seeded on a plastic surface which were cultured in said first culture medium devoid of any growth factor until the cells reached 85-90% confluency.

These cells were regularly phenotypically characterized by FACS (fluorescent cell sorting) or by any conventional means in order to detect the level of surface markers CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR.

When 95% of the cells express the positive surface markers CD73, CD90, CD105 and CD166 and less than 2% express the negative surface markers CD45, CD34 and HLA-DR, these cells are trypsinized and replated at a low density, e.g. 5000 MSC per cm ² in the second culture medium.

Preferably said " second culture medium " is any of the classic media commonly used to promote the growth of living primary cells. It may be the same as the "first culture medium", or it may be another medium selected from, for example, DMEM, DMEM/F12, MEM, alpha-MEM (α-MEM), IMDM or RPMI. More preferably, said second culture medium is DMEM (Dulbecco's Modified Eagle's Medium) or DMEM/F12 (Dulbecco's Modified Eagle's Medium: F-12 Nutritional Formula).

Even more preferably, said second culture medium contains serum or platelet lysate, for example 2-20% fetal calf serum and/or 1-5% platelet lysate. The most preferred second medium is DMEM containing 2-20% fetal bovine serum and 1-5% platelet lysate. It is also possible to use as a second culture medium a medium which is devoid of serum or platelet lysate, provided that it contains other suitable agents to promote the growth of living primary cells.

When the cells reach 40-50% confluence, pro-inflammatory growth factors or inflammatory mediators are added to the second culture medium and the cells are cultured in this medium until they reach 90-95% confluence.

Said "pro- inflammatory growth factors " are typically interleukins or chemokines known to have a pro-inflammatory effect. Examples of interleukins that can be added to the second culture medium include TNFα (tumor necrosis factor alpha), IL1 (interleukin 1), IL4, IL12, IL18 and IFNγ (interferon gamma). Examples of chemokines that can be added to the second culture medium include CXCL8, CXCL10, CXCL1, CXCL2, CXCL3, CCL2 and CCL5. Other inflammatory mediators (such as anti-inflammatory agents) may be used.

In a preferred embodiment, at least two pro-inflammatory growth factors are added to the second culture medium as defined above. These at least two pro-inflammatory growth factors are selected from the group consisting of TNFα, IL1, IL4, IL12, IL18 and IFNγ. In a more preferred embodiment, said pro-inflammatory growth factors are selected from IL1, IL4, IL12 and IL18. Even more preferably they are IL1 and IL4.

A typical concentration of growth factor(s) that can be added to MSC is 1-200 ng/ml, preferably 1-100 ng/ml, more preferably 10-80 ng/ml. Preferably, the step of culturing the MSC with the growth factor(s) lasts for at least one day, more preferably for two days.

The term "IL1" in this document refers to any isoform of interleukin 1, in particular, IL1α and IL1β. Isoforms of IL1 may be of different origins, depending on the intended use. For example, animal IL1 can be used for veterinary applications. Preferably, only IL1β is added to the second culture medium of the invention. In this particular embodiment, the concentration of added interleukin 1β may be 1-100 ng/ml, preferably 1-50 ng/ml, more preferably 10-40 ng/ml.

Reference to human IL1beta (IL1β) is given with accession number NP_000567.1 (SEQ ID NO: 6, 269 amino acids). The recombinant protein is commercially available under GMP conditions (R&D Systems, Thermofisher, Cellgenix, Peprotech).

The term "IL4" in this document means any isoform of interleukin 4. IL4 can be of different origins, depending on the intended use. For example, animal IL4 can be used for veterinary applications.

Reference to human IL4 is given with accession number AAA59149 (SEQ ID NO: 7, 153 amino acids). The recombinant protein is commercially available under GMP conditions (R&D Systems, Thermofisher, Cellgenix, Peprotech).

Any mixture of different pro-inflammatory growth factors can be used in the second medium of the invention. A particularly preferred embodiment is the use of a mixture of IL1 and IL4, more specifically IL1β and IL4, as disclosed in the experimental section below.

In this specific embodiment, the added interleukin 1β has a concentration in the second culture medium of 1-100 ng/ml, preferably 1-50 ng/ml, more preferably 10-40 ng/ml, and the added IL4 has a concentration of 1-100 ng /ml, preferably 1-50 ng/ml, more preferably 10-40 ng/ml. Preferably, the culture step with interleukin 1β and IL4 lasts for at least one day, more preferably for two days.

At the final stage, the cells are phenotypically characterized by any conventional means in order to reveal the level of surface markers CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR. These markers are well known in the art. All antibodies useful for detecting the level of expression of these markers are commercially available.

Expression of these cell surface markers can in particular be assessed using well known techniques such as cell membrane staining using biotinylation or other equivalent techniques, followed by immunoprecipitation using specific antibodies, flow cytometry, Western blot, ELISA (enzyme-linked immunosorbent assay). ) or ELISPOT (ELISA method), antibody microarrays or tissue microarrays combined with immunohistochemistry. Other suitable techniques include FRET (fluorescence resonant energy transfer) or BRET (bioluminescence resonant energy transfer), single cell microscopic or histochemical methods using single or multiple excitation wavelengths, and the use of any of the adapted optical methods such as electrochemical methods (techniques voltmetry and amperometry), atomic force microscopy and radio frequency based methods, e.g. resonance, ellipsometry, resonant mirror method, diffractive waveguide coupling or interferometry), cellular ELISA, radioisotope, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS -PAGE); HPLC (high performance liquid chromatography-mass spectrometry); liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS).

Preferably, cell surface marker levels are assessed by FACS. In other words, the method of the invention generally requires the following:

a) collection of mononuclear cells contained in perinatal biological tissue or fluid,

b) allowing said mononuclear cells to grow in the first culture medium until they reach a confluency of 85-90%, preferably on a plastic surface,

c) as soon as 95% of the cells express positive markers CD73, CD90, CD105 and CD166 and less than 2% express negative markers CD45, CD34 and HLA-DR, seed cells at a density of 1000 to 5000 MSC/cm2 in the ^second culture medium ,

d) adding 1-100 ng/ml of inflammatory mediators or pro-inflammatory growth factors once cells reach 40-50% confluence,

f) collection of cells when they reach 90-95% confluence.

The harvested cells can then be phenotypically characterized by FACS or any conventional means to detect the level of CD73, CD90, CD105, CD166, CD45, CD34 and HLA-DR surface markers. Said first and second culture media have been described above.

In step d) of said method, a typical concentration of added growth factor(s) is 1-200 ng/ml, preferably 1-100 ng/ml, more preferably 10-80 ng/ml. Preferably, the culturing step with the growth factor(s) lasts for at least one day, more preferably for two days.

In a specific embodiment of the invention, the concentration of added interleukin 1β or IL4 may be 1-100 ng/ml, preferably 1-50 ng/ml, more preferably 10-40 ng/ml. Preferably, the culture step with interleukin 1β and IL4 lasts for at least one day, more preferably for two days.

The final selected cells will be " Cell Culture of the Invention " or " CD106 ^High CD151 ⁺ Nestin ⁺ MSC of the Invention " or " MSC of the Invention ". This cell culture typically contains over 60%, preferably 60 to 70%, preferably over 70%, preferably over 80%, more preferably over 90% and even more preferably over 95% of cells expressing CD106. Moreover, it contains over 98%, preferably over 99% of cells expressing CD151. In addition, it contains over 98%, preferably over 99% of cells expressing nestin. Finally, it contains over 95%, preferably over 96%, preferably over 97%, preferably over 98% of cells expressing positive markers CD73, CD90, CD105 and CD166, and contains less than 2% of cells expressing negative markers CD45, CD34 and HLA-DR.

CD106 (also known as VCAM-1, “vascular endothelial adhesion protein type 1”) is known to have three isoforms. NP_001069.1 (herein referred to as SEQ ID NO: 1, 739 amino acids), NP_542413.1 (hereinafter referred to as SEQ ID NO: 2, 647 amino acids), and NP_001186763.1 (hereinafter referred to as SEQ ID NO: 3, 677 amino acids) are sequences of isoforms a, b and c, respectively. Antibodies to detect the level of expression of this particular biomarker are commercially available (eg Thermofisher, Abeam, OriGen, etc.). Expression of this marker on the surface of the cells of the invention is very important as it triggers pro-angiogenic activities that are essential for their therapeutic applications.

The biomarker nestin (referred to herein as SEQ ID NO: 4, 1621 amino acids) in humans is referenced as NP_006608.1. Antibodies to detect the level of expression of this particular biomarker are commercially available (eg, Thermofisher, Abeam, etc.).

The biomarker CD151 (referenced herein as SEQ ID NO: 5, 253 amino acids) in humans is referenced as NP_620599. Antibodies to detect the level of expression of this particular biomarker are commercially available (eg, Invitrogen, Sigma-Aldrich, Abeam, etc.).

In a preferred embodiment, the culture steps (or growth steps) are carried out on a plastic surface.

More specifically, the method of the invention may include the following steps:

a) possibly separate selection of placental tissues from multiple donors;

b) possibly washing the placental tissue three times with 1× PBS, dissecting into 1 mm ³ cubes and washing the tissue cubes again to remove most of the blood from this tissue;

c) it is possible to split the placental tissue of each donor separately with collagenase, centrifuge the split tissue and collect mononuclear cells;

d) inoculation of the selected mononuclear cells in the culture medium;

e) trypsinization and passage of cells as soon as they reach 85-90% confluence;

f) characterizing these cells based on the percentage of cells that express positive markers CD73, CD90, CD105 and CD166 and negative markers CD45, CD34 and HLA-DR;

e) seeding cells in culture medium containing 90% Dulbecco's Modified Eagle's/Knockout F12 (DMEM/F12-KO), 10% FBS and growth factors at a seeding density of 1000 to 5000 MSC/cm ² when they contain at least 95% positive markers and at most 2% negative markers;

h) adding 1-100 ng/ml interleukin 1β and optionally 1-100 ng/ml IL4 when they are 40-50% confluent;

i) trypsinizing and harvesting the cells once they reach 90-95% confluency; and

j) it is possible to characterize cells based on the percentage of cells that express positive markers CD73, CD90, CD105 and CD166 and negative markers CD45, CD34 and HLA-DR.

The results disclosed below show that CD106 expression is significantly upregulated on the surface of MSCs obtained by the method of the invention either from the umbilical cord or from the placenta. This expression is very important for the therapeutic use of cells thus obtained (see below).

The present application therefore also relates to a method for increasing the CD106 expression level of undifferentiated MSCs, said method comprising culturing a population of undifferentiated MSCs under the specific conditions carefully described above, all detailed embodiments being applied mutatis mutandis.

In another aspect, this invention relates to a cell culture obtained by the above method. This cell culture, as a rule, contains isolated CD106 high CD151 ⁺ Nestin ⁺ MSC, expressing the marker - vascular endothelial adhesion molecule type 1 (VCAM-1) at a detectably higher level than mesenchymal stem cells derived from adult bone marrow, adipose tissue, umbilical cord or placenta that were not in contact with any growth factors at the time they were received.

In a preferred embodiment, the cell culture of the invention is characterized in that:

(i) over 60%, preferably over 70%, more preferably over 80%, and even more preferably over 90% of the cells express CD106 at a detectable level, and

(ii) over 98%, preferably over 99% of the cells express CD151 at a detectable level, and

(iii) over 90%, preferably over 92%, more preferably over 95% of the cells express nestin at a detectable level, and

(iv) over 95%, preferably over 96%, preferably over 97%, preferably over 98% of the cells express positive markers CD73, CD90, CD105 and CD166 at a detectable level.

In a more preferred embodiment, said cell culture is characterized in that it contains over 98% of MSCs that do not express the CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104 and CD133 markers at a detectable level. These markers are well known in the art and antibodies that detect them are commercially available.

In a preferred embodiment, the cell culture of the invention contains:

- over 60%, preferably over 70%, more preferably over 80% and even more preferably over 90% of cells expressing CD106 at a detectable level, and

- over 98%, preferably over 99% of cells expressing CD151 at a detectable level, and

- over 98%, preferably over 99% of cells expressing nestin at a detectable level, and

- over 95%, preferably over 96%, preferably over 97%, preferably over 98% of cells expressing CD73, CD90, CD105 and CD166 markers at a detectable level, and

- less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level.

According to the present invention, a cell " expresses a marker at a detectable level " if said marker is present on its surface at a significant level, i. if the signal associated with the staining of the specified surface marker (typically obtained with an antibody that recognizes the specified marker, and the specified antibody, for example, associated with a fluorescent dye), which is measured for the specified cell, exceeds the signal corresponding to the staining of a single cell known not to express the specified marker. The specialist is well aware of how to identify these cells/markers, so that these protocols do not need to be described in detail here.

The results disclosed in the experimental part of the present application show that the cell culture obtained by the method of the invention is capable of inducing angiogenesis in vitro and in vivo . These results further demonstrate that administration of said cells to individuals (human or animal subjects) suffering from an ischemic disease or a circulatory disorder results in a detectable improvement in one or more symptoms of said disease or disorder.

The results disclosed below also show that the methods of the invention are capable of producing cells expressing high levels of the CD106/VCAM1 membrane protein. Importantly, this protein has recently been associated with the expression of pro-angiogenic cytokines. Consequently, CD106+ MSCs were chosen for their pro-angiogenic efficacy and proposed as a treatment for hind limb ischemia ([16], [17]).

Therefore, in a third aspect, the invention relates to the use of said cells as a medicament for the treatment of an individual suffering from an ischemic disease or a disorder of the circulatory system. In other words, the present invention relates to the use of said cells for the manufacture of a medicament for use in the treatment of subjects suffering from an ischemic disease or a disorder of the circulatory system. The medicament of the invention may also be applied to the vascular capillary network of the skin and may include dermatological and cosmetic applications.

Any mammal can be treated with the cells of the invention. Said mammal may be a domestic animal (dog, cat, horse, etc.) or a bovine animal (sheep, goat, cow, etc.). It will be apparent to those skilled in the art that when an animal is to be treated according to the method of the invention, the original undifferentiated MSCs will be obtained from a biological sample from the same animal species (allogeneic graft) or from a similar species (heterologous graft) and the growth factors that are used in the second culture medium will correspond to the growth factors of the same animal species. For example, if a cat is to be treated, then the original MSCs will be obtained from the perinatal tissue or body fluid of the cat, and feline IL1β (recombinant or non-recombinant) will be added to the second culture medium, possibly along with feline IL4.

In a preferred embodiment, said mammal is a human. In this case, the parent MSCs will be derived from perinatal tissue or from a woman's body fluid and human IL1β (recombinant or non-recombinant, e.g. SEQ ID NO: 6) will be added to the second culture medium, possibly along with human IL4 (e.g., SEQ ID NO: 7).

To this end, the cell culture of the invention may be transplanted or applied topically to said subject by any conventional means. In this case, the present invention is directed to a method for treating a subject suffering from ischemic disease, circulatory system disorder, immunopathology, organ damage or organ dysfunction, said method comprising the step of transplanting the cell culture described above into said subject. This transplantation can be carried out using an implanted reservoir, or by in situ injection of cells into the muscle, or by intravenous injection, or by any suitable delivery system. This application can also be carried out topically, by directly bringing the cells into contact with the skin or mucosa, or by applying the cells to the skin or any mucosa by a device, or by delivering the cells to the skin or mucosa by any suitable delivery system.

Preferably said disease or disorder is selected from the group consisting of the following: type I diabetes mellitus, type II diabetes, GVHD (graft versus host disease), aplastic anemia, multiple sclerosis, Duchenne muscular dystrophy, rheumatoid arthritis, stroke, idiopathic pulmonary fibrosis, dilated cardiomyopathy, osteoarthritis, cirrhosis, liver failure, renal failure, peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease, heart failure, diabetic ulcer or any degenerative disease, synechia, endometrial disorder or fibrotic disorder of the gastrointestinal tract, such as anal fistula. More preferably, said disease or disorder is peripheral arterial occlusive disease, critical limb ischemia, peripheral vascular disease, or diabetic ulcer. In a specific embodiment, said disease or disorder is a skin or mucosal disease, including diabetic ulcer, ulcer, trauma, burn, hot liquid burn, wound or wound healing problem, bed sore, wart, etc. (but not limited to them).

The cell culture according to the invention can in particular be used in a dermatological preparation, the purpose of which is the treatment of skin pathologies such as burns, wounds, ulcers, scars, warts, or other diseases such as synechia or fibrotic disorders of the gastrointestinal tract (for example, anal fistula).

In another specific embodiment, said disease or disorder is anal fistula or endometrial injury.

The present application covers other uses. In particular, it is possible to use the MSC according to the invention for dermatological or cosmetic purposes, for example, to regenerate skin or mucosal cells, improve the appearance of the skin or mucosa, correct a defect in the skin or mucosa, or treat a burnt area of the skin or mucosa.

In order to increase efficacy and facilitate administration of a drug of the invention, the cell culture of the invention may be mixed with any agent, composition of agents, or other biocompatible substance or device. The cell culture of the invention may also be encapsulated or incorporated into any suitable delivery system or biocompatible substance. These cells or a preparation containing these cells can be applied using a medical device such as, for example, an endoscope, stent, or syringe. It can also be applied topically by bringing the cells into contact with the skin or mucosa.

The present invention is also directed to a medical device containing the cell culture of the invention. The term "medical device" in this document covers any device, apparatus, tool, machine, device, implant, reagent for the introduction of a therapeutic composition. In the context of the present invention, said medical device is, for example, a patch, a stent, an endoscope or a syringe.

The present invention is also directed to a delivery system containing the cell culture of the invention. The term "delivery system" in this document covers any system (environment or carrier) for the introduction of a pharmaceutical product to a patient. It may be an oral delivery system or a controlled release system. In the context of the present invention, said delivery system is, for example, liposomes, proliposomes, microspheres, micro- or nanovesicles of biopolymers, lipids or nanoparticles.

In a preferred embodiment of the invention, the cells of the invention are incorporated into a hydrogel or other biocompatible substance or excipient. Said hydrogel may include, in particular, sodium alginate hydrogel, hyaluronic acid hydrogel, chitosan hydrogel, collagen hydrogel, HPMC (hydroxypropyl methylcellulose) hydrogel, poly-L-lysine hydrogel, poly-L-glutamic acid hydrogel, polyvinyl alcohol (PVA) hydrogel, polyacrylic acid hydrogel, polymethacrylic acid hydrogel, polyacrylamide (PAM) hydrogel, and poly-N-acrylamide (PNAM) hydrogel.

The present invention also relates to a hydrogel containing the MSC of the invention and optionally another biocompatible substance or excipient. An alginate hydrogel is preferred herein, such as the alginate hydrogel described in CN 106538515.

In the context of the present invention, " biocompatible substances " are substances classically used in biomedical applications. They are, for example, metals (such as stainless steel, cobalt alloys, titanium alloys), ceramics (aluminum oxide, zirconium dioxide, calcium phosphates), polymers (silicones, poly(ethylene), poly(vinyl chloride), polyurethanes, polylactides) or natural polymers (alginate, collagen, gelatin, elastin, etc.). These substances can be synthetic or natural. Biocompatible excipients are well known in the art and therefore need not be described in detail.

This hydrogel can be used for cosmetic or therapeutic purposes.

The present invention also relates to a pharmaceutical or veterinary composition containing the cell culture of the invention, as well as its use in the treatment of diseases and disorders mentioned above. It also relates to a dermatological or cosmetic composition containing a cell culture according to the invention.

This pharmaceutical, veterinary or cosmetic composition may additionally contain other biocompatible agents (eg hydrogel) as described above.

The specified composition preferably contains at least about 1 to 5×10 ⁶ cells.

DESCRIPTION OF GRAPHICS

In order for this disclosure to be easily understood and practiced, reference will now be made to exemplary embodiments and the illustrated drawings. These drawings, together with the detailed description below, serve to further illustrate embodiments and explain the various principles and advantages of the present disclosure.

On FIG. 1 describes a diagram showing how the CD106 ^high CD151 ⁺ Nestin ⁺ MSC subset of the invention can be obtained.

On FIG. 2 shows the morphology of MSCs attached to culture flasks in the placenta according to Example 1.

On FIG. 3 shows adipogenic and osteogenic cell differentiation CD106 ^high CD151 ⁺ nestin ⁺ MSC. These results indicate that the CD106 ^high CD151 ⁺ Nestin ⁺ MSC subpopulation is capable of adipogenic and osteogenic differentiation in the appropriate environment. White arrows highlight lipid droplets and a highly mineralized area.

On FIG. 4 shows pathological examination by angiography: representative angiograms obtained on postoperative day 21.

On FIG. 5 shows pathological examination by LDPI: representative LDPI images obtained on postoperative day 0 and day 21. In the panel above, the area is still affected 3 weeks after transplantation, as indicated by the white arrow. In the lower panels, black arrows highlight the perfused area 3 weeks after transplantation.

On FIG. 6 shows the results of infusion of placental-derived MSCs in diabetic patients. A total of 15 patients with diabetes (11 men and 4 women) were included in this clinical trial. (A) shows the reduction in mean insulin requirement after 3x treatment with CD106 ^high CD151 ⁺ Nestin ⁺ MSC derived from perinatal tissue in this cohort of 15 patients, which was statistically significant (P < 0.001). (B) shows that treatment with CD106 ^high CD151 ⁺ Nestin ⁺ MSC of perinatal origin also significantly improved glycated hemoglobin levels in diabetic patients (P < 0.001). These results indicate that CD106 ^high CD151 ⁺ Nestin ⁺ MSC of perinatal origin may be a good choice for diabetic patients.

Fig. 7. (A) shows the level of ascites, determined using ultrasound, before infusion of CD106 ^high CD151 ⁺ Nestin ⁺ MSC in a patient suffering from decompensated cirrhosis. And (B) shows the level of ascites, determined using ultrasound, one month after the introduction of CD106 ^high CD151 ⁺ Nestin ⁺ MSC. (C) shows that the liver functions of said patient improved significantly. CA125 expression decreased to normal levels. In addition, total protein, albumin, and serum globulin also increased significantly.

On FIG. 8 shows perfusion rate evaluation in a mouse NOD/SCID model of hindlimb ischemia after arterial ligation and injection of various doses of umbilical cord MSC of the invention or saline or VEGF (vascular endothelial growth factor).

EXAMPLES

For simplicity and illustrative purposes, the present invention is described by reference to its typical embodiments. However, one of ordinary skill in the art will appreciate that the present invention may be practiced without being limited to these specific details. In other instances, well-known methods have not been described in detail in a manner that does not unnecessarily obscure the present invention.

1. Method for obtaining MSC according to the invention from placental tissue

The method according to the invention was carried out by the following:

a) separate sampling of human placental tissue from multiple donor mothers;

b) washing this placental tissue three times with 1x PBS, dissecting into 1 mm ³ cubes and washing the cube tissue again to remove most of the blood from this tissue;

c) splitting the placental tissue of each donor separately with collagenase, centrifuging the split tissue and collecting mononuclear cells;

d) inoculation of these mononuclear cells in the culture medium;

e) trypsinization and passage of cells as soon as they reach 85-90% confluence;

f) characterizing these cells based on the percentage of cells that express positive markers CD73, CD90, CD105 and CD166 and negative markers CD45, CD34 and HLA-DR;

g) seeding cells in culture medium containing 90% Dulbecco's Modified Eagle's Medium/Knockout F12 (DMEM/F12-KO), 10% FBS and growth factors at a seeding density of 1000 to 5000 MSC/cm ² when they contain at least 95% positive markers and at most 2% negative markers;

h) obtaining CD106 ^high CD151 ⁺ Nestin ⁺ MSC containing over 80% of cells that express CD106, 98% for CD151, 98% for nestin and 95% of cells that express positive markers CD73, CD90, CD105 and CD166, and less, than 2% of cells that express CD45, CD34 and HLA-DR negative markers (for transplantation use), and the addition of 20 ng/ml IL1β (provided by Peprotech) and 20 ng/ml IL4 (provided by Peprotech) when they are confluent at 40 -fifty%;

i) trypsinizing and harvesting the cells once they reach 90-95% confluency;

j) cell characterization based on the percentage of cells that express CD73, CD90, CD105 and CD166 positive markers and CD45, CD34 and HLA-DR negative markers.

This method provided a subpopulation of MSCs of placental origin with a yield of at least 1x10 ⁵ cells/cm ² MSC (5x10 ⁶ MSCs in T75 vials when 90% confluent) that can be used in allogeneic administrations. These cell cultures contained over 95% of cells that express positive markers CD73, CD90, CD105 and CD166 and less than 2% of cells that express negative markers CD45, CD34 and HLA-DR.

Human perinatal tissue-derived CD106 ^high CD151 ⁺ Nestin ⁺ MSC were fibroblast-like and they grow very well on a plastic vial (cf. Fig. 2).

Conducted three independent experiments (experiment 1, experiment 2 and experiment 3). Added 20 ng/ml and interleukin 1 (β), and 4, when the cells were 40-50% confluent (see Fig. 1). Currently, MSCs comprise over 95% of cells that express positive markers CD73, CD90, CD105 and CD166 and less than 2% of cells that express negative markers CD45, CD34 and HLA-DR. Thereafter, the MSCs were kept growing until 90% of the cells were confluent (see Tables 1 and 2 below), in practice for about 2 days.

Notably, the expression of the cell surface protein MSC CD106, CD151, and nestin was significantly increased after the addition of interleukins 1 and 4.

TABLE 1 shows the expression of CD11b, CD19, CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD106, CD151, nestin and HLA-DR on placental tissue-derived MSCs before adding interleukin 1 and 4 obtained in Examples 1, 2 and 3 with placenta. These results indicate that placenta-derived MSCs have classical cell surface markers, just like bone marrow- and adipose-derived MSCs.

TABLE 2 shows the expression of CD11b, CD19, CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD106, CD151, nestin and HLA-DR on placental tissue-derived MSCs after addition of interleukins 1 and 4 in Experiments 1, 2 and 3. These results show that the protein markers CD106 and nestin were significantly increased 48 hours after the addition of IL-1 and IL-4 in complete medium. Meanwhile, CD106 ^high CD151 ⁺ nestin ⁺ cells still have the classic phenotypic cell surface marker MSC.

These subpopulations may secrete additional growth factors, cytokines, immunomodulatory factors, and inflammatory factors (see Table 3). Data CD106 ^high CD151 ⁺ Nestin ⁺ MSC will therefore have a better potential for immunomodulation and angiogenesis.

Table 3 shows the associated levels of expression of growth factors secreted into the depleted nutrient medium, determined by qPCR (quantitative polymerase chain reaction). These results indicate that placental tissue-derived MSCs have greater protein expression or secretion of IL-6, IL-8, IL-10, HGF, ANG, MMP2, VEGF-A, and TGF-β 48 hours after IL addition. -1 and IL-4 in full environment.

2. Method for obtaining MSC according to the invention from umbilical cord tissues

In this example, the following reagents were used:

2.1. Isolation of cells by explant method

The umbilical cord was removed from the transport solution and cut into lengths of 2-3 cm. In order to avoid contamination by adhering blood cells, each umbilical cord segment containing a blood clot that could not be removed was discarded. These sections were then disinfected in a vessel with antibiotics and antifungal agents containing αMEM plus 1 g/l vancomycin plus 1 g/l amoxicillin plus 500 mg/l amikacin plus 50 mg/l amphotericin B for 30 min at room temperature (RT) . Antibiotics were immediately dissolved in sterile water for injection.

The umbilical cord segments were removed from the vessel and washed quickly in 1x PBS at RT. The epithelial membrane was slightly incised without touching the vessels. Each segment was then cut into pieces of 0.5 cm thickness and placed in the bottom of a 150 cm ² pastmass vial with a cap. 6 to 10 cuts per vial were placed with a free space circle of at least 1 cm radius around each piece and allowed to attach for 15 minutes without medium at RT.

After attachment, complete medium (αMEM plus 5% CPL plus 2 U/ml heparin) was carefully added, keeping the explants attached to the bottom of the vial. These vials were then incubated at 37°C, 90% humidity and 5% CO ₂ .

The culture medium was replaced after 5-7 days.

On day 10 after isolation, cell migration from the explants was monitored by inverted microscopy. If a circle of adherent cells was visible around most of the explants, they were carefully removed by pulling out of the vial through the cap using a pair of sterile disposable tweezers.

Starting from this stage, cell confluence was visually checked every other day, and, if necessary, the medium was changed on day 17.

When the cells reached 70-90% confluence or at C20 (day 20), the medium was removed and the cells were washed with 30 ml of 1x PBS per vial. Cells were then removed using Trypzean®, harvested with old medium and centrifuged for 10 minutes at 2500 rpm. The supernatant was discarded and then the cells were suspended in a cryopreservation solution consisting of αMEM plus 100 mg/ml HSA (human serum albumin) plus 10% DMSO (dimethyl sulfoxide) and cryopreserved.

2.2. Isolation of cells by enzymatic method

The umbilical cord was removed from the transport solution and cut into lengths of 2-3 cm. In order to avoid contamination with adhering blood cells, each section containing a blood clot that could not be removed was discarded. These sections were then disinfected in a vessel with antibiotics and antifungals containing αDMEM plus 1 g/l vancomycin plus 1 g/l amoxicillin plus 500 mg/l amikacin plus 50 mg/l amphotericin for 30 minutes at room temperature (RT). Antibiotics were immediately dissolved in sterile water for injection.

The umbilical cord was then cut into small pieces, immersed in an enzyme mixture containing 2.7 mg/ml collagenase type I and 0.7 mg/ml hyaluronidase, incubated for 3 hours at 37°C with gentle shaking, followed by the addition of 2.5 % trypsin and additional incubation for 30 min.

The split suspension was diluted 1:2 with medium to reduce the viscosity of the suspension and passed through a nylon mesh to obtain a single cell suspension. Cells were centrifuged at 300 g for 20 min and seeded at a density of 10,000 cells/cm ² using fresh medium.

The culture medium was replaced after 5-7 days and every 7 days thereafter.

When the cells reached 70-90% confluence or at C20, the medium was removed and the cells were washed with 30 ml of 1x PBS per vial. Cells were then removed using Trypzean®, harvested with old medium and centrifuged for 10 minutes at 2500 rpm. The supernatant was discarded and then the cells were suspended in a cryopreservation solution consisting of αMEM plus 100 mg/ml HSA plus 10% DMSO and cryopreserved.

2.3. Thawing and cell culture

Cells were thawed following the classical protocol. Briefly, the cryovials were removed from liquid nitrogen and quickly immersed in a 37°C water bath. v/v) ciprofloxacin plus 2 U/ml heparin plus 5% (v/v) LP) and centrifuged rapidly (300 g, RT, 5 min).

After centrifugation, cells were suspended in prewarmed complete medium and evaluated for number and viability (trypan blue/Mallassez hemocytometer).

Cells were seeded in two 75 cm ² plastic culture flasks in complete medium and incubated (90% humidity, 5% CO ₂ , 37°C).

2.4. cell stimulation

After several days of expansion, the cells were checked for confluency. When confluency reached 30-50%, the old medium was discarded and replaced with either fresh complete unstimulated medium or fresh medium supplemented with 10 ng/ml IL-1β and 10 ng/ml IL-4.

Cells were then incubated for at least 2 days before experiments using flow cytometry.

2.5. Cell collection and analysis by flow cytometry

After 2-3 days of expansion/stimulation, the cells were checked for confluency. If the confluence was up to 80%, the cells were harvested. Briefly, the old medium was discarded and the cells were washed with 1x DPBS. Trypsin EDTA (ethylenediaminetetraacetic acid) was added and the cells were incubated for 5 min at 37°C. Trypsin was neutralized with at least 2x the medium volume, and the cell suspension was collected and evaluated for cell number and viability.

The flow cytometry experiment required 1×10 ⁶ cells, which were centrifuged and resuspended in 1× DPBS plus 0.4% HSA.

Cells were labeled for CD73, CD90, CD105, CD106, CD151 and CD31, CD34, CD45, HLA-DR according to the following protocol:

1. Cells were evaluated for viability and number.

2. The required volume of suspension to obtain 20,000 cells/labeling tube was placed in a 15 ml propylene plastic tube.

3. This tube was centrifuged for 5 min at 300 g and 4°C.

4. Cells were resuspended using 1× DPBS plus 0.4% HSA (diluted 10 times with 4 mg/ml HSA in 1× DPBS). The required volume was 500 µl per labeling tube.

Extracellular staining was performed for CD106 and CD151 markers as recommended by the FACS manufacturer and antibody suppliers. Spent intracellular staining for nestin. For intracellular staining, a fixation/permeabilization solution referred to as "BD Cytofix/Cytoperm kit" was used.

If the cells were not analyzed immediately after staining, a fixation step was performed by bringing the cells into contact with a solution of 1x DPBS/0.5% formaldehyde.

After labeling, cells were washed with 1x DPBS plus 0.4% HSA and permeabilized to label nestin.

Labeled cells were analyzed with a BD Biosciences Accuri C6+ Cytometer and the results were analyzed using the BD Accuri C6 Plus software.

2.6. results

Several batches of umbilical cord-derived MSCs (HB-COR001 to COR005-MSC) were obtained by culturing umbilical cord MSCs isolated by the explant methods described above (2.1.) under the conditions disclosed in paragraph 2.4. above. For umbilical cord 3 and 5 (COR003 and COR005), two MSC populations (MSC1 and MSC2) were concomitantly obtained by repeating the steps of the invention in a separate manner.

The expression of CD106 on the surface of these cells was measured by flow cytometry.

All batches of MSC tested showed a dramatic increase (greater than 60%) in CD106 expression levels (see Table 4).

3. Effect of the isolation protocol on the expression of CD106 MSCs originating from the umbilical cord

Mesenchymal stem cells were isolated from the umbilical cord using the two different methods described above (see 2.1. and 2.2.): by isolation of explants and by enzymatic digestion.

All cells were cultured in the same culture medium (αMEM plus 5% platelet lysate (LP)), stimulated during passage 3 according to the method of the invention, and harvested 2 days after stimulation. Cell viability is measured and cells are counted.

The expression of several phenotypic markers, including CD106, was then measured by flow cytometry.

All MSCs, whatever the isolation method or stimulation conditions, expressed phenotypic markers in a classical manner: over 95% are positive for CD73, CD90, CD105 while they were negative for CD31, CD34, CD45 and HLA-DR.

For both CD151+ and nestin, over 95% were also positive, regardless of stimulation or isolation method.

Without stimulation, MSCs isolated by enzymatic digestion expressed higher levels of CD106 prior to stimulation (53%) than MSCs isolated using explant methods (20%). However, they were less sensitive to stimulation without the inflammatory mixture: they observed a smaller increase in CD106 (an increase of plus 4% compared to an increase of plus 60%).

The explant method is therefore the preferred method of the invention for cord-derived MSCs.

4. Effects of stimulation with a combination of IL1-IL4 compared with stimulation with IL1 or IL4 alone on umbilical cord mesenchymal stem cell CD106 levels

Mesenchymal stem cells were isolated from two umbilical cords by explant isolation (see 2.1.).

All cells were cultured in the same culture medium (αMEM plus 5% platelet lysate (LP)), stimulated at passage 4 according to the method of the invention with a mixture of 10 ng/ml IL-1b and 10 ng/ml IL-4, or each interleukin alone ( 10 ng/ml each) and harvested 2 days after stimulation. Cell viability is measured and cells are counted.

The expression of several phenotypic markers, including CD106, was then measured by flow cytometry.

All MSCs, regardless of stimulation conditions, expressed phenotypic markers in a classical manner: over 95% are positive for CD73, CD90, CD105, while they were negative for CD31, CD34, CD45 and HLA-DR. Over 95% were also CD151+ positive regardless of stimulation.

With regard to the CD106 marker, the increase in expression observed using stimulation with a combination of IL1 and IL4 is 3-5 times higher than the increase in expression observed with stimulation with interleukin alone (see Table 7).

5. Effect of MSC CD106 neovascularization ^high CD151 ⁺ Nestin ⁺ placental origin in diabetic mice

This example focuses on the effect of therapeutic neovascularization of MSC CD106 ^high CD151 ⁺ Nestin ⁺ placental origin. It provides a deep understanding of their potential for clinical application as a cell-based therapy, in combination with insulin injection for the treatment of critical hind limb ischemia in diabetes. The results of the inventors showed that MSC CD106 ^high CD151 ⁺ Nestin ⁺ originating from the placenta are involved in angiogenesis and therapeutic vascularization in order to improve ischemic state and restore blood flow perfusion through direct differentiation into vascular cells. In addition, MSC CD106 ^high placenta-derived CD151 ⁺ Nestin ⁺ improved ischemic injury and functional recovery in diabetic rats.

Immunodeficient male nude rats at six weeks of age were purchased from Vital River Laboratories (supplier of Charles River Laboratories in China). Diabetes was induced by a single intraperitoneal injection of streptozotocin (70 mg/kg in citrate buffer solution, prepared just before injection) after an overnight fast. Fasting plasma glucose levels were measured every week, and rats with plasma glucose levels between 11 mM and 15 mM were considered diabetic. Age and weight matched nude rats receiving intraperitoneal injection of citrate buffer were used as non-diabetic controls (glycemia 5.5 to 8 mM).

Two weeks later, diabetic nude rats were anesthetized (60 mg/kg pentobarbital ip) and the left femoral artery was occluded with a 3-0 silk ligature. This ligature was applied 0.5 cm proximal to the bifurcation of the saphenous and popliteal arteries. Lipiodol (1.5 ml/kg) was used to induce intravascular embolism. A mock ligature was applied to the left femoral artery, leaving the left hind limb non-ischemic.

Once the ischemia pattern was established, the surgical insufficiency of the limb was evident as the limb could no longer bear any weight and the paw was swollen and red. Over time, this ischemic injury improved to varying degrees in all groups. Limb function recovery was significantly increased in the CD106 ^high CD151 ⁺ Nestin ⁺ MSC subpopulation (Fig. 4).

More specifically, the histological data in FIG. 4 show that capillary density increased in the cell transplant groups compared to the PBS group. In addition, more capillaries were observed in the CD106 ^high CD151 ⁺ Nestin ⁺ MSC subpopulation cell transplant groups in diabetic rats and non-diabetic rats compared to the PBS group.

On FIG. 5 shows functional evidence of ischemia-induced changes using LDPI vascularization. This image shows that blood flow was completely blocked in the left hind paw on the day of surgery; this is indicated by a deep dark color. After three weeks, blood flow perfusion recovered to some extent in all groups, but did not return to normal in the PBS group, where the ischemic/non-ischemic perfusion ratio only reached 52%. Perfusion recovery in the cell transplant groups was significantly greater. This ratio was 81% in the prior art P-MSC group (P less than 0.05 compared to the PBS group) and 86% for the CD106 ^high CD151 ⁺ Nestin ⁺ MSC subpopulation group (P less than 0.01 compared to the PBS group) .

Perfusion recovery in both MSC subpopulations was significantly greater. Therefore, these results revealed that MSCs improve blood flow perfusion and that the MSCs of the invention are more effective in doing so. In fact, the MSCs of the invention showed a significant improvement in blood flow restoration in both diabetic and non-diabetic rats.

These results revealed that the MSCs of the invention are more effective in improving blood flow perfusion than the MSCs of the prior art. This example shows that the introduction of MSC according to the invention is a promising new approach against diabetic critical limb ischemia.

6. Therapeutic effect of MSC CD106 ^high CD151 ⁺ Nestin ⁺ originating from the placenta in patients with diabetes

This example focuses on the therapeutic effect of MSC CD106 ^high CD151 ⁺ Nestin ⁺ originating from the placenta, in patients suffering from diabetes. It provides a deep understanding of their potential for clinical applications as cell-based therapies for diabetes.

A total of 15 patients with diabetes (11 men and 4 women) were included in this clinical study. Inclusion criteria were patients with type 2 diabetes diagnosed between November 2013 and November 2014 at Tianjin General Hospital. These patients were aged 30 to 85 years, had diabetes for 3 years or more, required insulin for optimal glycemic control at a dose of 0.7 U/kg/day or more for at least 1.5 years, had insulin dysfunction, poorly controlled fluctuations in blood glucose during insulin-based treatment and willingness to participate in this study. These 15 patients ranged in age from 42 to 67 years with a median age of 59 years; duration of diabetes ranged from 3 years to 17 years with a median of 8 years; daily insulin requirement ranged from 38 units to 90 units with an average of 58.7 units. Glucose tolerance testing, insulin release testing, C-peptide stimulation testing, and glycated hemoglobin testing were performed simultaneously every three months. Conducted analyzes of the function of the heart, liver and kidney. During treatment, adverse events and side effects were observed. It was considered effective if the daily insulin requirement was reduced by 50% or more after treatment, and this effect lasted more than 3 months.

Patients received three intravenous infusions of the placenta-derived MSC (P-MSC) of the invention with a one-month infusion interval. The total amount of P-MSC administered to each patient was (1.0-1.5)×10 ⁶ /kg with a mean value of 1.25×10 ⁶ /kg. At the same time, patients continued to use insulin and adjusted their insulin dose according to their blood glucose levels. In terms of complications, the original general treatment was maintained. All patients were followed up after therapy for at least 6 months.

This clinical trial showed a reduction in mean insulin requirement after 3 injections of the placenta-derived MSC according to the invention in this group of 15 patients, which was statistically significant (P less than 0.001). The injectable insulin dosage for diabetic patients was reduced by almost half after administration of the placenta-derived MSCs of the invention (compared to the mean insulin dosage taken over the 6 months prior to MSC administration) (FIG. 6A).

This clinical trial showed that placental-derived MSC treatment according to the invention significantly reduced the level of glycated hemoglobin in diabetic patients (P less than 0.001) (FIG. 6B).

These data confirm that the MSC of the invention can be used to treat diabetic patients.

7. Therapeutic effect of the placenta-derived MSC according to the invention in patients with aplastic anemia

This example focuses on the therapeutic effect of the placenta-derived MSCs of the invention in patients with aplastic anemia and provides a thorough understanding of their potential for clinical use as a cell-based therapy for aplastic anemia.

Aplastic anemia is mainly regarded as an immune-mediated bone marrow insufficiency (BM) syndrome characterized by hypoplasia and pancytopenia with fatty BM and reduced angiogenesis. Previous studies have demonstrated that acquired aplastic anemia is manifested by abnormalities in the HSC (hematopoietic stem cell)/HPC (hematopoietic progenitor cell) and hematopoietic microenvironment. Plenty of evidence has hinted that aplastic anemia may be a syndrome characterized by stem/progenitor cell disorders, including HSC/HPC and MSC. MSCs support hematopoiesis and regulate almost all immune cell functions to maintain hematopoietic and immune homeostasis. MSCs can modulate the functions of major immune cells, including T cells, B cells, monocytes, DCs (dendritic cells), NKTs, and neutrophils [5]. MSCs have remarkable immunosuppressive properties against Th1, Th17 and CTL. MSCs inhibit T cell proliferation, IFN-γ and TNF-α secretion by Th1 cells, while stimulating Th2 cells to produce IL-10 and proliferate Treg cells. However, recent studies have shown that MSCs from patients with aplastic anemia have poor proliferation and incomplete MLR immunosuppression, PHA-induced T cell activation, and IFN-γ release [6,7]. A recent study by the inventors showed that MSCs from patients with aplastic anemia were attenuated in suppressing the proliferation and clonogenic potential of CD4+ T cells while promoting Treg cell proliferation. It was also found that MSCs were defective in suppressing the production of TNF-α and IFN-γ by CD4+ cells. However, there was no significant difference in the regulation of IL-4, IL-10 and IL-17 production [8]. In addition, the inventors' study also showed that MSCs from aplastic anemia patients showed abnormal morphology, reduced proliferation and clonogenic potential, and increased apoptosis compared to BM-MSCs from healthy controls. MSCs from patients with aplastic anemia were sensitive to induction to differentiate into adipocytes, but more difficult to differentiate into osteoblasts. Consistent with abnormal biological characteristics, a large number of genes involved in the cell cycle, cell division, proliferation, chemotaxis, and hematopoietic cell line showed markedly reduced expression in MSCs from these aplastic anemia patients. Conversely, more genes associated with apoptosis, adipogenesis and immune response showed increased expression in MSCs from patients with aplastic anemia. The MSC gene expression profile further confirmed abnormal biological properties and provided significant evidence for a possible mechanism for disruption of the VM microenvironment in aplastic anemia [9].

MSCs represent a promising therapeutic candidate for the treatment of aplastic anemia due to the following two important facts:

i) in general, supporting hematopoiesis and potent immunosuppressive capacity of MSC and

ii) the difference in biological characteristics and functional deficiency observed in MSCs derived from patients with aplastic anemia.

In a clinical trial, a 6-year-old girl with intermittent fever lasting for more than a month was treated with MSC. Aplastic anemia was confirmed after two bone marrow biopsies revealed hypocellularity. After treatment with cyclosporine and stanozolol for almost 6 months, there was no improvement in the peripheral blood phenotype. Hematopoietic stem cell transplant was not a good choice as it was very difficult to find a suitable donor. Therefore, this patient was given an intravenous infusion of 1×10 ⁷ placenta-derived MSC according to the invention. The peripheral blood phenotype significantly improved after the 1st MSC transplant. However, this patient was still dependent on transfusions of blood products, including red blood cells and platelets. After 6 months, this patient received a 2nd time intravenous injection of 1×10 ⁷ MSC according to the invention. The peripheral blood phenotype improved significantly and reached almost normal levels 12 months after the 2nd injection. In addition, this patient was completely transfusion free of blood products 12 months after the 2nd injection (Table 8). These data confirm that the MSCs derived from the placenta of the invention can be used for the clinical treatment of patients with aplastic anemia.

8. Therapeutic effect of the placenta-derived MSCs of the invention in patients with liver diseases

This example focuses on the therapeutic effect of placenta-derived MSCs prepared according to the method of the invention (ie with IL1 and IL4 treatment) administered to patients suffering from liver diseases. It provides a deep understanding of their potential for clinical applications as cell-based therapies for liver diseases.

In this clinical trial, a 40-year-old man was confirmed to have suffered from alcoholic hepatitis for more than ten years. It was confirmed that this patient had decompensated cirrhosis two years ago, although he received conventional treatment. Since there is no good choice for the treatment of decompensated cirrhosis, this patient voluntarily received an intravenous injection of 4×10 ⁷ MSC according to the invention.

In this clinical trial, the results of the ultrasound examination showed an ascites level of 9.7 cm before MSC injection (see Fig. 7A). However, the ascites level decreased to 3.6 cm one month after MSC administration (see Fig. 7B). In addition, these results showed that the liver function of the patient with decompensated cirrhosis improved significantly after administration of MSC. In addition, serum CA125 expression decreased to normal levels according to clinical controls.

These results demonstrate an excellent clinical response in patients with decompensated cirrhosis treated with the MSC of the invention.

In addition, total protein, albumin, and serum globulin also increased significantly, reaching normal levels according to clinical reference values (FIG. 7B). These results showed that the synthetic and metabolic function of the patient's liver improved one month after the intravenous injection of MSC.

Therefore, the placenta-derived MSCs of the invention represent a very good cytotherapeutic choice against liver diseases.

9. Angiogenic potential of umbilical cord-derived cells of the invention

The pro-angiogenic potential of the MSCs of the invention was further analyzed in a mouse model of hind limb ischemia.

Material and methods

Twelve 8 week old NOD/SCID mice (Laboratoire Janvier) were anesthetized with a mixture of ketamine and xylazine. The left proximal and distal femoral artery of the left leg was then ligated (silk suture 6.0, Ethicon, Issy-Les-Moulineaux, France) and the part between the applied ligatures was excised. Mice that show more than 80% ischemia after surgery were intramuscularly injected into the gastrocnemius muscle of the ischemic limb with 2 doses of the MSC of the invention prepared according to the protocols disclosed in paragraph 2.4 above (0.5×10 ⁶ cells per animal in group 2, 0.05×10 ⁶ cells per animal in group 3). Saline injection was used in control group 1. Control group 4 was also injected with a dose of 20 ng/ml VEGF/mouse.

The injection schedule as well as the dose was for the intended clinical use of the cells, and the dose administered to the animals was more than 10 times the suggested dose for the Phase I/II human clinical trial (which is about 25×10 ⁶ cells/kg, which means about 0.5×10 ⁶ cells/mouse (average mouse weight is 20 g)).

Rear limb blood flow was measured using a laser scanning Doppler setup (Laser Doppler perfusion system, Perimed PeriScan PIM III). Mean perfusion of ischemic and non-ischemic limbs was determined before and after ischemia, and every 7 days until 21 days after ischemia. Changes in laser frequency dependent on blood flow were visualized using different colored pixels. The images were analyzed to quantify blood flow using a blood perfusion analysis program (LPDIwin). The percentage of perfusion was expressed as the ratio of ischemic to non-ischemic hind limb.

Staining sections of the muscles of the hind limb with hematoxylin-eosin

Mice were sacrificed at the end of the experiment (C21). The gastrocnemius muscles of the hind limb were dissected and stained to visualize the distribution of cell nuclei in the muscle fiber. Muscles isolated from an ischemic hind limb were fixed in paraffin and stained with hematoxylin/eosin.

results

On FIG. 8 shows mean reperfusion after hindlimb ischemia in mice treated with different injections: 0.9% NaCl (negative control - n is 8), 20 ng/ml VEGF (positive control - n is 6), 50 x 10 ³ cells MS (n is 9) and 500×10 ³ MS cells (n is 7).

These results demonstrated that administration of high doses of the MSC of the invention to mice after femoral artery ligation resulted in greater reperfusion rates compared to vehicle (NaCl) or VEGF alone. A 100% reperfusion rate was observed as early as 7 days after cell injection for mice receiving the highest cell dose (0.5×10 ⁶ cells). There was a statistically significant difference with other groups.

A dose-response effect is also likely because in this preliminary experiment, the highest cell dose (0.5×10 ⁶ cells) appears to achieve a better perfusion score than a 10-fold lower dose.

These results confirm the actual angiogenic activity of the MSCs of the invention in improving blood perfusion in this CLI mouse model.

Results observed with hematoxylin-eosin staining (data not shown):

In normal, non-ischemic muscles, cell nuclei are distributed at the periphery of the muscle fiber.

In ischemic mice from groups 1 (0.9% NaCl) and 4 (20 ng/ml VEGF) 21 days after induction of ischemia, cells show very few nuclei at the periphery of the muscle fiber as they are still regenerating.

In ischemic muscles of mice from group 3 (0.05×10 ⁶ MSC cells or 50,000) 21 days after induction of ischemia, 50% of the muscle fibers are regenerated and show nuclei not at the periphery of the fiber. Regeneration is faster than in groups 1 and 4.

In ischemic muscles of mice from group 2 (0.5×10 ⁶ MSC cells or 500,000) 21 days after induction of ischemia, 100% of the muscle fibers are regenerated and show nuclei not at the periphery of the fiber. The staining profile of the muscle is identical to normal non-ischemic muscle. In this experiment, a dose of 500,000 cells provides complete muscle regeneration.

BIBLIOGRAPHICAL REFERENCES

1. Shi M, Zhang Z, Xu R, Lin H, Fu J, Zou Z, Zhang A, Shi J, Chen L, Lv S et ah Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients. Stem cells translational medicine 2012, 1 (10):725-731.

2. Wang L, Wang L, Cong X, Liu G, Zhou J, Bai B, Li Y, Bai W, Li M, Ji H et al : Human umbilical cord mesenchymal stem cell therapy for patients with active rheumatoid arthritis: safety and efficacy. Stem cells and development 2013, 22 (24):3192-3202.

3. Rodrigo SF, van Ramshorst J, Hoogslag GE, Boden H, Velders MA, Cannegieter SC, Roelofs H, Al Younis I, Dibbets-Schneider P, Fibbe WE et al : Intramyocardial injection of autologous bone marrow-derived ex vivo expanded mesenchymal stem cells in acute myocardial infarction patients is feasible and safe up to 5 years of follow-up. Journal of cardiovascular translational research 2013, 6 (5):816-825.

4. Gupta PK, Chullikana A, Parakh R, Desai S, Das A, Gottipamula S, Krishnamurthy S, Anthony N, Pherwani A, Majumdar AS: A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia. Journal of translational medicine 2013, 11 :143.

5. Chen X, Armstrong MA, Li G: Mesenchymal stem cells in immunoregulation. Immunology and cell biology 2006, 84 (5):413-421.

6. Bacigalupo A, Valle M, Podesta M, Pitto A, Zocchi E, De Flora A, Pozzi S, Luchetti S, Frassoni F, Van Lint MT et al : T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Experimental hematology 2005, 33 (7):819-827.

7. Chao YH, Peng CT, Harn HJ, Chan CK, Wu KH: Poor potential of proliferation and differentiation in bone marrow mesenchymal stem cells derived from children with severe aplastic anemia. Annals of Hematology 2010, 89 (7):715-723.

8. Li J, Lu S, Yang S, Xing W, Feng J, Li W, Zhao Q, Wu H, Ge M, Ma F et al : Impaired immunomodulatory ability of bone marrow mesenchymal stem cells on CD4(+) T cells in aplastic anemia. Results in immunology 2012, 2 :142-147.

9. Li J, Yang S, Lu S, Zhao H, Feng J, Li W, Ma F, Ren Q, Liu B, Zhang L et al : Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia. PloS one 2012, 7 (11):e47764.

10. Anna Otte, Vesna Bucan, Kerstin Reimers, and Ralf Hass. Mesenchymal Stem Cells Maintain Long-Term In Vitro Sternness During Explant Culture. Tissue Engineering Part C: Methods. November 2013.19(12): 937-948

11. Van Pham, P., Truong, N.C., Le, P.TB. et al. Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications. Cell Tissue Bank (2016) 17:289

12. Dong Li et al, Biological characteristics of human placental mesenchymal stem cells and their proliferative response to various cytokines. Cells Tissues Organs 2007; 186:169-179.

13. Abomaray FM et al, Phenotypic and functional characterization of mesenchymal stem/multipotent Stromal cells from Decidua Basalis of human term placenta. Stem cells international , volume 2016, Article ID5184601

14. Savilova AM et al, Comparaison of the expression of immunomodulatory factors in cultures of mesenchymal stromal cells from human extraembryonic tissues . Cell Technologies in Biology and Medicine, №4, February 2015

15. Hongye Fan et al, Pre-treatment with IL-1β enhances the efficacy of MSC transplantation in DSS-induced colitis. Cellular and Molecular Immunology (2012), 9, 473-481

16. Han ZC, et al, New insights into the heterogeneity and functional diversity of human mesenchymal stem cells. Biomedical Materials and Engineering 28(2017) S29-S45

17. Du W. et al, VCAM-1+ placenta chorionic villi-derived mesenchymal stem cells display potent pro-angiogenic activity. Stem Cell research & therapy (2016) 7:49

--->

SEQUENCE LIST

<110> HEALTH AND BIOTECH FRANCE

<120> MESENCHYMAL STEM

CELLS: METHOD FOR THEIR PRODUCTION AND APPLICATION

<130> B373499 D36728

<150> PCT/IB/2016/001936

<151> 2016-12-12

<160> 7

<170>PatentIn version 3.5

<210> 1

<211> 739

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> Human CD106 isoform

<400> 1

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp

1 5 10 15

Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu

20 25 30

Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser

35 40 45

Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp

50 55 60

Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu

65 70 75 80

Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr

85 90 95

Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile

100 105 110

Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu

115 120 125

Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro

130 135 140

Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys

145 150 155 160

Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys

165 170 175

Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val

180 185 190

Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro

195 200 205

Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys

210 215 220

Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly

225 230 235 240

Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile

245 250 255

Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly

260 265 270

Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile

275 280 285

Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val

290 295 300

Glu Leu Ile Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser Pro Gly

305 310 315 320

Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr Cys Ser

325 330 335

Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln Ile Asp

340 345 350

Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser Thr Leu

355 360 365

Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu Cys Thr

370 375 380

Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val Glu Leu

385 390 395 400

Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly Leu Val

405 410 415

Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro

420 425 430

Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile Leu Glu

435 440 445

Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu Asn Lys

450 455 460

Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly Lys Ala

465 470 475 480

Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe Glu Pro

485 490 495

Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala Pro Arg

500 505 510

Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu Gly Ser

515 520 525

Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro Lys Ile

530 535 540

Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu Ser Glu

545 550 555 560

Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser Gly Val

565 570 575

Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys Glu Val

580 585 590

Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr Ala Phe

595 600 605

Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser Cys Thr

610 615 620

Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys Ala Glu

625 630 635 640

Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr Ile Arg

645 650 655

Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser Lys Asn

660 665 670

Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln Gly Arg

675 680 685

Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu Tyr Phe

690 695 700

Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr Phe Ala

705 710 715 720

Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys

725 730 735

Ser Lys Val

<210> 2

<211> 647

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> human CD106 isoform b

<400> 2

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp

1 5 10 15

Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu

20 25 30

Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser

35 40 45

Thr Thr Gly Cys Glu Ser Pro Phe Phe Ser Trp Arg Thr Gln Ile Asp

50 55 60

Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu

65 70 75 80

Thr Met Asn Pro Val Ser Phe Gly Asn Glu His Ser Tyr Leu Cys Thr

85 90 95

Ala Thr Cys Glu Ser Arg Lys Leu Glu Lys Gly Ile Gln Val Glu Ile

100 105 110

Tyr Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro Leu Glu

115 120 125

Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val Tyr Pro

130 135 140

Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu Met Lys

145 150 155 160

Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu Thr Lys

165 170 175

Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly Lys Val

180 185 190

Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser Val Pro

195 200 205

Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser Pro Lys

210 215 220

Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu Gly Gly

225 230 235 240

Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro Glu Ile

245 250 255

Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu Ser Gly

260 265 270

Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser Gly Ile

275 280 285

Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys Glu Val

290 295 300

Glu Leu Ile Val Gln Ala Phe Pro Arg Asp Pro Glu Ile Glu Met Ser

305 310 315 320

Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro

325 330 335

Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu

340 345 350

Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser

355 360 365

Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp

370 375 380

Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met

385 390 395 400

Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn

405 410 415

Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu

420 425 430

Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro

435 440 445

Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln

450 455 460

Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu

465 470 475 480

Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser

485 490 495

Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys

500 505 510

Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile

515 520 525

Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys

530 535 540

Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala

545 550 555 560

Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys

565 570 575

Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp

580 585 590

Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu

595 600 605

Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile

610 615 620

Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val

625 630 635 640

Glu Ala Gln Lys Ser Lys Val

645

<210> 3

<211> 677

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> isoform c human CD106

<400> 3

Met Pro Gly Lys Met Val Val Ile Leu Gly Ala Ser Asn Ile Leu Trp

1 5 10 15

Ile Met Phe Ala Ala Ser Gln Ala Phe Lys Ile Glu Thr Thr Pro Glu

20 25 30

Ser Arg Tyr Leu Ala Gln Ile Gly Asp Ser Val Ser Leu Thr Cys Ser

35 40 45

Thr Thr Gly Ser Phe Pro Lys Asp Pro Glu Ile His Leu Ser Gly Pro

50 55 60

Leu Glu Ala Gly Lys Pro Ile Thr Val Lys Cys Ser Val Ala Asp Val

65 70 75 80

Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu Leu Lys Gly Asp His Leu

85 90 95

Met Lys Ser Gln Glu Phe Leu Glu Asp Ala Asp Arg Lys Ser Leu Glu

100 105 110

Thr Lys Ser Leu Glu Val Thr Phe Thr Pro Val Ile Glu Asp Ile Gly

115 120 125

Lys Val Leu Val Cys Arg Ala Lys Leu His Ile Asp Glu Met Asp Ser

130 135 140

Val Pro Thr Val Arg Gln Ala Val Lys Glu Leu Gln Val Tyr Ile Ser

145 150 155 160

Pro Lys Asn Thr Val Ile Ser Val Asn Pro Ser Thr Lys Leu Gln Glu

165 170 175

Gly Gly Ser Val Thr Met Thr Cys Ser Ser Glu Gly Leu Pro Ala Pro

180 185 190

Glu Ile Phe Trp Ser Lys Lys Leu Asp Asn Gly Asn Leu Gln His Leu

195 200 205

Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala Met Arg Met Glu Asp Ser

210 215 220

Gly Ile Tyr Val Cys Glu Gly Val Asn Leu Ile Gly Lys Asn Arg Lys

225 230 235 240

Glu Val Glu Leu Ile Val Gln Glu Lys Pro Phe Thr Val Glu Ile Ser

245 250 255

Pro Gly Pro Arg Ile Ala Ala Gln Ile Gly Asp Ser Val Met Leu Thr

260 265 270

Cys Ser Val Met Gly Cys Glu Ser Pro Ser Phe Ser Trp Arg Thr Gln

275 280 285

Ile Asp Ser Pro Leu Ser Gly Lys Val Arg Ser Glu Gly Thr Asn Ser

290 295 300

Thr Leu Thr Leu Ser Pro Val Ser Phe Glu Asn Glu His Ser Tyr Leu

305 310 315 320

Cys Thr Val Thr Cys Gly His Lys Lys Leu Glu Lys Gly Ile Gln Val

325 330 335

Glu Leu Tyr Ser Phe Pro Arg Asp Pro Glu Ile Glu Met Ser Gly Gly

340 345 350

Leu Val Asn Gly Ser Ser Val Thr Val Ser Cys Lys Val Pro Ser Val

355 360 365

Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu Leu Lys Gly Glu Thr Ile

370 375 380

Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr Asp Met Lys Ser Leu Glu

385 390 395 400

Asn Lys Ser Leu Glu Met Thr Phe Ile Pro Thr Ile Glu Asp Thr Gly

405 410 415

Lys Ala Leu Val Cys Gln Ala Lys Leu His Ile Asp Asp Met Glu Phe

420 425 430

Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr Leu Tyr Val Asn Val Ala

435 440 445

Pro Arg Asp Thr Thr Val Leu Val Ser Pro Ser Ser Ile Leu Glu Glu

450 455 460

Gly Ser Ser Val Asn Met Thr Cys Leu Ser Gln Gly Phe Pro Ala Pro

465 470 475 480

Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln Pro Leu

485 490 495

Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser Thr Lys Met Glu Asp Ser

500 505 510

Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln Ala Gly Arg Ser Arg Lys

515 520 525

Glu Val Glu Leu Ile Ile Gln Val Thr Pro Lys Asp Ile Lys Leu Thr

530 535 540

Ala Phe Pro Ser Glu Ser Val Lys Glu Gly Asp Thr Val Ile Ile Ser

545 550 555 560

Cys Thr Cys Gly Asn Val Pro Glu Thr Trp Ile Ile Leu Lys Lys Lys

565 570 575

Ala Glu Thr Gly Asp Thr Val Leu Lys Ser Ile Asp Gly Ala Tyr Thr

580 585 590

Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly Val Tyr Glu Cys Glu Ser

595 600 605

Lys Asn Lys Val Gly Ser Gln Leu Arg Ser Leu Thr Leu Asp Val Gln

610 615 620

Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser Pro Glu Leu Leu Val Leu

625 630 635 640

Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala Ile Gly Met Ile Ile Tyr

645 650 655

Phe Ala Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala

660 665 670

Gln Lys Ser Lys Val

675

<210> 4

<211> 1621

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> Human nestin

<400> 4

Met Glu Gly Cys Met Gly Glu Glu Ser Phe Gln Met Trp Glu Leu Asn

1 5 10 15

Arg Arg Leu Glu Ala Tyr Leu Ala Arg Val Lys Ala Leu Glu Glu Gln

20 25 30

Asn Glu Leu Leu Ser Ala Glu Leu Gly Gly Leu Arg Ala Gln Ser Ala

35 40 45

Asp Thr Ser Trp Arg Ala His Ala Asp Asp Glu Leu Ala Ala Leu Arg

50 55 60

Ala Leu Val Asp Gln Arg Trp Arg Glu Lys His Ala Ala Glu Val Ala

65 70 75 80

Arg Asp Asn Leu Ala Glu Glu Leu Glu Gly Val Ala Gly Arg Cys Gln

85 90 95

Gln Leu Arg Leu Ala Arg Glu Arg Thr Thr Glu Glu Val Ala Arg Asn

100 105 110

Arg Arg Ala Val Glu Ala Glu Lys Cys Ala Arg Ala Trp Leu Ser Ser

115 120 125

Gln Val Ala Glu Leu Glu Arg Glu Leu Glu Ala Leu Arg Val Ala His

130 135 140

Glu Glu Glu Arg Val Gly Leu Asn Ala Gln Ala Ala Cys Ala Pro Arg

145 150 155 160

Cys Pro Ala Pro Pro Arg Gly Pro Pro Ala Pro Ala Pro Glu Val Glu

165 170 175

Glu Leu Ala Arg Arg Leu Gly Glu Ala Trp Arg Gly Ala Val Arg Gly

180 185 190

Tyr Gln Glu Arg Val Ala His Met Glu Thr Ser Leu Gly Gln Ala Arg

195 200 205

Glu Arg Leu Gly Arg Ala Val Gln Gly Ala Arg Glu Gly Arg Leu Glu

210 215 220

Leu Gln Gln Leu Gln Ala Glu Arg Gly Gly Leu Leu Glu Arg Arg Ala

225 230 235 240

Ala Leu Glu Gln Arg Leu Glu Gly Arg Trp Gln Glu Arg Leu Arg Ala

245 250 255

Thr Glu Lys Phe Gln Leu Ala Val Glu Ala Leu Glu Gln Glu Lys Gln

260 265 270

Gly Leu Gln Ser Gln Ile Ala Gln Val Leu Glu Gly Arg Gln Gln Leu

275 280 285

Ala His Leu Lys Met Ser Leu Ser Leu Glu Val Ala Thr Tyr Arg Thr

290 295 300

Leu Leu Glu Ala Glu Asn Ser Arg Leu Gln Thr Pro Gly Gly Gly Ser

305 310 315 320

Lys Thr Ser Leu Ser Phe Gln Asp Pro Lys Leu Glu Leu Gln Phe Pro

325 330 335

Arg Thr Pro Glu Gly Arg Arg Leu Gly Ser Leu Leu Pro Val Leu Ser

340 345 350

Pro Thr Ser Leu Pro Ser Pro Leu Pro Ala Thr Leu Glu Thr Pro Val

355 360 365

Pro Ala Phe Leu Lys Asn Gln Glu Phe Leu Gln Ala Arg Thr Pro Thr

370 375 380

Leu Ala Ser Thr Pro Ile Pro Pro Thr Pro Gln Ala Pro Ser Pro Ala

385 390 395 400

Val Asp Ala Glu Ile Arg Ala Gln Asp Ala Pro Leu Ser Leu Leu Gln

405 410 415

Thr Gln Gly Gly Arg Lys Gln Ala Pro Glu Pro Leu Arg Ala Glu Ala

420 425 430

Arg Val Ala Ile Pro Ala Ser Val Leu Pro Gly Pro Glu Glu Pro Gly

435 440 445

Gly Gln Arg Gln Glu Ala Ser Thr Gly Gln Ser Pro Glu Asp His Ala

450 455 460

Ser Leu Ala Pro Pro Leu Ser Pro Asp His Ser Ser Leu Glu Ala Lys

465 470 475 480

Asp Gly Glu Ser Gly Gly Ser Arg Val Phe Ser Ile Cys Arg Gly Glu

485 490 495

Gly Glu Gly Gln Ile Trp Gly Leu Val Glu Lys Glu Thr Ala Ile Glu

500 505 510

Gly Lys Val Val Ser Ser Leu Gln Gln Glu Ile Trp Glu Glu Glu Asp

515 520 525

Leu Asn Arg Lys Glu Ile Gln Asp Ser Gln Val Pro Leu Glu Lys Glu

530 535 540

Thr Leu Lys Ser Leu Gly Glu Glu Ile Gln Glu Ser Leu Lys Thr Leu

545 550 555 560

Glu Asn Gln Ser His Glu Thr Leu Glu Arg Glu Asn Gln Glu Cys Pro

565 570 575

Arg Ser Leu Glu Glu Asp Leu Glu Thr Leu Lys Ser Leu Glu Lys Glu

580 585 590

Asn Lys Glu Leu Leu Lys Asp Val Glu Val Val Arg Pro Leu Glu Lys

595 600 605

Glu Ala Val Gly Gln Leu Lys Pro Thr Gly Lys Glu Asp Thr Gln Thr

610 615 620

Leu Gln Ser Leu Gln Lys Glu Asn Gln Glu Leu Met Lys Ser Leu Glu

625 630 635 640

Gly Asn Leu Glu Thr Phe Leu Phe Pro Gly Thr Glu Asn Gln Glu Leu

645 650 655

Val Ser Ser Leu Gln Glu Asn Leu Glu Ser Leu Thr Ala Leu Glu Lys

660 665 670

Glu Asn Gln Glu Pro Leu Arg Ser Pro Glu Val Gly Asp Glu Glu Ala

675 680 685

Leu Arg Pro Leu Thr Lys Glu Asn Gln Glu Pro Leu Arg Ser Leu Glu

690 695 700

Asp Glu Asn Lys Glu Ala Phe Arg Ser Leu Glu Lys Glu Asn Gln Glu

705 710 715 720

Pro Leu Lys Thr Leu Glu Glu Glu Asp Gln Ser Ile Val Arg Pro Leu

725 730 735

Glu Thr Glu Asn His Lys Ser Leu Arg Ser Leu Glu Glu Gln Asp Gln

740 745 750

Glu Thr Leu Arg Thr Leu Glu Lys Glu Thr Gln Gln Arg Arg Arg Ser

755 760 765

Leu Gly Glu Gln Asp Gln Met Thr Leu Arg Pro Pro Glu Lys Val Asp

770 775 780

Leu Glu Pro Leu Lys Ser Leu Asp Gln Glu Ile Ala Arg Pro Leu Glu

785 790 795 800

Asn Glu Asn Gln Glu Phe Leu Lys Ser Leu Lys Glu Glu Ser Val Glu

805 810 815

Ala Val Lys Ser Leu Glu Thr Glu Ile Leu Glu Ser Leu Lys Ser Ala

820 825 830

Gly Gln Glu Asn Leu Glu Thr Leu Lys Ser Pro Glu Thr Gln Ala Pro

835 840 845

Leu Trp Thr Pro Glu Glu Ile Asn Gln Gly Ala Met Asn Pro Leu Glu

850 855 860

Lys Glu Ile Gln Glu Pro Leu Glu Ser Val Glu Val Asn Gln Glu Thr

865 870 875 880

Phe Arg Leu Leu Glu Glu Glu Asn Gln Glu Ser Leu Arg Ser Leu Gly

885 890 895

Ala Trp Asn Leu Glu Asn Leu Arg Ser Pro Glu Glu Val Asp Lys Glu

900 905 910

Ser Gln Arg Asn Leu Glu Glu Glu Glu Asn Leu Gly Lys Gly Glu Tyr

915 920 925

Gln Glu Ser Leu Arg Ser Leu Glu Glu Glu Gly Gln Glu Leu Pro Gln

930 935 940

Ser Ala Asp Val Gln Arg Trp Glu Asp Thr Val Glu Lys Asp Gln Glu

945 950 955 960

Leu Ala Gln Glu Ser Pro Pro Gly Met Ala Gly Val Glu Asn Glu Asp

965 970 975

Glu Ala Glu Leu Asn Leu Arg Glu Gln Asp Gly Phe Thr Gly Lys Glu

980 985 990

Glu Val Val Glu Gln Gly Glu Leu Asn Ala Thr Glu Glu Val Trp Ile

995 1000 1005

Pro Gly Glu Gly His Pro Glu Ser Pro Glu Pro Lys Glu Gln Arg

1010 1015 1020

Gly Leu Val Glu Gly Ala Ser Val Lys Gly Gly Ala Glu Gly Leu

1025 1030 1035

Gln Asp Pro Glu Gly Gln Ser Gln Gln Val Gly Ala Pro Gly Leu

1040 1045 1050

Gln Ala Pro Gln Gly Leu Pro Glu Ala Ile Glu Pro Leu Val Glu

1055 1060 1065

Asp Asp Val Ala Pro Gly Gly Asp Gln Ala Ser Pro Glu Val Met

1070 1075 1080

Leu Gly Ser Glu Pro Ala Met Gly Glu Ser Ala Ala Gly Ala Glu

1085 1090 1095

Pro Gly Pro Gly Glyn Gly Val Gly Gly Leu Gly Asp Pro Gly His

1100 1105 1110

Leu Thr Arg Glu Glu Val Met Glu Pro Pro Leu Glu Glu Glu Ser

1115 1120 1125

Leu Glu Ala Lys Arg Val Gln Gly Leu Glu Gly Pro Arg Lys Asp

1130 1135 1140

Leu Glu Glu Ala Gly Gly Leu Gly Thr Glu Phe Ser Glu Leu Pro

1145 1150 1155

Gly Lys Ser Arg Asp Pro Trp Glu Pro Pro Arg Glu Gly Arg Glu

1160 1165 1170

Glu Ser Glu Ala Glu Ala Pro Arg Gly Ala Glu Glu Ala Phe Pro

1175 1180 1185

Ala Glu Thr Leu Gly His Thr Gly Ser Asp Ala Pro Ser Pro Trp

1190 1195 1200

Pro Leu Gly Ser Glu Glu Ala Glu Glu Asp Val Pro Pro Val Leu

1205 1210 1215

Val Ser Pro Ser Pro Thr Tyr Thr Pro Ile Leu Glu Asp Ala Pro

1220 1225 1230

Gly Pro Gln Pro Gln Ala Glu Gly Ser Gln Glu Ala Ser Trp Gly

1235 1240 1245

Val Gln Gly Arg Ala Glu Ala Leu Gly Lys Val Glu Ser Glu Gln

1250 1255 1260

Glu Glu Leu Gly Ser Gly Glu Ile Pro Glu Gly Pro Gln Glu Glu

1265 1270 1275

Gly Glu Glu Ser Arg Glu Glu Ser Glu Glu Asp Glu Leu Gly Glu

1280 1285 1290

Thr Leu Pro Asp Ser Thr Pro Leu Gly Phe Tyr Leu Arg Ser Pro

1295 1300 1305

Thr Ser Pro Arg Trp Asp Pro Thr Gly Glu Gln Arg Pro Pro Pro

1310 1315 1320

Gln Gly Glu Thr Gly Lys Glu Gly Trp Asp Pro Ala Val Leu Ala

1325 1330 1335

Ser Glu Gly Leu Glu Ala Pro Pro Ser Glu Lys Glu Glu Gly Glu

1340 1345 1350

Glu Gly Glu Glu Glu Cys Gly Arg Asp Ser Asp Leu Ser Glu Glu

1355 1360 1365

Phe Glu Asp Leu Gly Thr Glu Ala Pro Phe Leu Pro Gly Val Pro

1370 1375 1380

Gly Glu Val Ala Glu Pro Leu Gly Gln Val Pro Gln Leu Leu Leu

1385 1390 1395

Asp Pro Ala Ala Trp Asp Arg Asp Gly Glu Ser Asp Gly Phe Ala

1400 1405 1410

Asp Glu Glu Glu Ser Gly Glu Glu Gly Glu Glu Asp Gln Glu Glu

1415 1420 1425

Gly Arg Glu Pro Gly Ala Gly Arg Trp Gly Pro Gly Ser Ser Val

1430 1435 1440

Gly Ser Leu Gln Ala Leu Ser Ser Ser Gln Arg Gly Glu Phe Leu

1445 1450 1455

Glu Ser Asp Ser Val Ser Val Ser Val Pro Trp Asp Asp Ser Leu

1460 1465 1470

Arg Gly Ala Val Ala Gly Ala Pro Lys Thr Ala Leu Glu Thr Glu

1475 1480 1485

Ser Gln Asp Ser Ala Glu Pro Ser Gly Ser Glu Glu Glu Ser Asp

1490 1495 1500

Pro Val Ser Leu Glu Arg Glu Asp Lys Val Pro Gly Pro Leu Glu

1505 1510 1515

Ile Pro Ser Gly Met Glu Asp Ala Gly Pro Gly Ala Asp Ile Ile

1520 1525 1530

Gly Val Asn Gly Gln Gly Pro Asn Leu Glu Gly Lys Ser Gln His

1535 1540 1545

Val Asn Gly Gly Val Met Asn Gly Leu Glu Gln Ser Glu Glu Val

1550 1555 1560

Gly Glyn Gly Met Pro Leu Val Ser Glu Gly Asp Arg Gly Ser Pro

1565 1570 1575

Phe Gln Glu Glu Glu Gly Ser Ala Leu Lys Thr Ser Trp Ala Gly

1580 1585 1590

Ala Pro Val His Leu Gly Gln Gly Gln Phe Leu Lys Phe Thr Gln

1595 1600 1605

Arg Glu Gly Asp Arg Glu Ser Trp Ser Ser Gly Glu Asp

1610 1615 1620

<210> 5

<211> 253

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> human CD151

<400> 5

Met Gly Glu Phe Asn Glu Lys Lys Thr Thr Cys Gly Thr Val Cys Leu

1 5 10 15

Lys Tyr Leu Leu Phe Thr Tyr Asn Cys Cys Phe Trp Leu Ala Gly Leu

20 25 30

Ala Val Met Ala Val Gly Ile Trp Thr Leu Ala Leu Lys Ser Asp Tyr

35 40 45

Ile Ser Leu Leu Ala Ser Gly Thr Tyr Leu Ala Thr Ala Tyr Ile Leu

50 55 60

Val Val Ala Gly Thr Val Val Met Val Thr Gly Val Leu Gly Cys Cys

65 70 75 80

Ala Thr Phe Lys Glu Arg Arg Asn Leu Leu Arg Leu Tyr Phe Ile Leu

85 90 95

Leu Leu Ile Ile Phe Leu Leu Glu Ile Ile Ala Gly Ile Leu Ala Tyr

100 105 110

Ala Tyr Tyr Gln Gln Leu Asn Thr Glu Leu Lys Glu Asn Leu Lys Asp

115 120 125

Thr Met Thr Lys Arg Tyr His Gln Pro Gly His Glu Ala Val Thr Ser

130 135 140

Ala Val Asp Gln Leu Gln Gln Glu Phe His Cys Cys Gly Ser Asn Asn

145 150 155 160

Ser Gln Asp Trp Arg Asp Ser Glu Trp Ile Arg Ser Gln Glu Ala Gly

165 170 175

Gly Arg Val Val Pro Asp Ser Cys Cys Lys Thr Val Val Ala Leu Cys

180 185 190

Gly Gln Arg Asp His Ala Ser Asn Ile Tyr Lys Val Glu Gly Gly Cys

195 200 205

Ile Thr Lys Leu Glu Thr Phe Ile Gln Glu His Leu Arg Val Ile Gly

210 215 220

Ala Val Gly Ile Gly Ile Ala Cys Val Gln Val Phe Gly Met Ile Phe

225 230 235 240

Thr Cys Cys Leu Tyr Arg Ser Leu Lys Leu Glu His Tyr

245 250

<210> 6

<211> 269

<212> PRT

<213> homo sapiens

<220>

<221> other_characteristic

<223> human IL1beta

<400> 6

Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser

1 5 10 15

Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln Met

20 25 30

Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp Gly Gly Ile

35 40 45

Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys Gly Phe Arg Gln Ala

50 55 60

Ala Ser Val Val Val Ala Met Asp Lys Leu Arg Lys Met Leu Val Pro

65 70 75 80

Cys Pro Gln Thr Phe Gln Glu Asn Asp Leu Ser Thr Phe Phe Pro Phe

85 90 95

Ile Phe Glu Glu Glu Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala

100 105 110

Tyr Val His Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp

115 120 125

Ser Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala

130 135 140

Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met

145 150 155 160

Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro Val Ala Leu

165 170 175

Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp

180 185 190

Lys Pro Thr Leu Gln Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys

195 200 205

Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys Ile Glu Ile Asn Asn

210 215 220

Lys Leu Glu Phe Glu Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr

225 230 235 240

Ser Gln Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly

245 250 255

Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser

260 265

<210> 7

<211> 153

<212> PRT

<213> homo sapiens

<220>

<221> MISC_FEATURE

<223> human IL4

<400> 7

Met Gly Leu Thr Ser Gln Leu Leu Pro Pro Leu Phe Phe Leu Leu Ala

1 5 10 15

Cys Ala Gly Asn Phe Val His Gly His Lys Cys Asp Ile Thr Leu Gln

20 25 30

Glu Ile Ile Lys Thr Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys

35 40 45

Thr Glu Leu Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr

50 55 60

Glu Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr

65 70 75 80

Ser His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln Gln

85 90 95

Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg Leu Asp Arg

100 105 110

Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys Pro Val Lys Glu Ala

115 120 125

Asn Gln Ser Thr Leu Glu Asn Phe Leu Glu Arg Leu Lys Thr Ile Met

130 135 140

Arg Glu Lys Tyr Ser Lys Cys Ser Ser

145 150

<---

Claims (90)

1. A method for obtaining CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC), which includes culturing a population of undifferentiated MSC from placental and extraembryonic tissues in a culture medium containing a mixture of IL1β and IL4. 2. The method of claim 1, wherein said undifferentiated MSCs are derived from a biological tissue selected from the placenta, umbilical cord, or placental membranes, or components thereof, or from a biological fluid such as cord blood, placental blood, or amniotic fluid. 3. The method according to any one of paragraphs. 1 and 2, where these undifferentiated MSCs are obtained by isolating cells from explant tissues. 4. The method according to any one of paragraphs. 1-3, wherein said undifferentiated MSCs are obtained from the umbilical cord, preferably by isolating cells from an umbilical cord fragment explant. 5. The method according to any one of paragraphs. 1-4, wherein said undifferentiated MSCs are obtained from the placenta, preferably by isolating cells from a piece of placental tissue. 6. The method according to any one of paragraphs. 1-5, where the specified culture medium contains from 1 to 100 ng/ml of interleukin 1β. 7. The method according to any one of paragraphs. 1-6, where the specified culture medium contains from 1 to 100 ng/ml of interleukin 4. 8. A method for increasing the expression level of CD106 undifferentiated MSCs, comprising cultivating a population of undifferentiated MSCs from placental and extraembryonic tissues in a culture medium containing a mixture of IL1β and IL4. 9. The method of claim 8, wherein said undifferentiated MSCs are derived from a biological tissue selected from the placenta, umbilical cord, or placental membranes, or components thereof, or from a biological fluid such as cord blood, placental blood, or amniotic fluid. 10. The method according to any one of paragraphs. 8, 9, wherein said undifferentiated MSCs are obtained by isolating cells from tissue explants. 11. The method according to any one of paragraphs. 8-10, wherein said undifferentiated MSCs are obtained from the umbilical cord, preferably by isolating cells from an umbilical cord fragment explant. 12. The method according to any one of paragraphs. 8-11, wherein said undifferentiated MSCs are obtained from the placenta, preferably by isolating cells from a piece of placental tissue. 13. The method according to any one of paragraphs. 8-12, in which the specified culture medium contains from 1 to 100 ng/ml of interleukin 1, preferably interleukin 1β. 14. The method according to any one of paragraphs. 8-13, in which the specified culture medium contains from 1 to 100 ng/ml of interleukin 4. 15. Cell culture for the treatment of diabetes containing CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) obtained by the method according to paragraphs. 1-14, wherein said cell culture contains - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level. 16. Cell culture for the treatment of aplastic anemia containing CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) obtained by the method according to paragraphs. 1-14, wherein said cell culture contains - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level. 17. Cell culture for the treatment of decompensated liver cirrhosis containing CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) obtained by the method according to paragraphs. 1-14, wherein said cell culture contains - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level. 18. Cell culture for the induction of angiogenesis containing CD106 ^high CD151 ⁺ Nestin ⁺ mesenchymal stem cells (MSC) obtained by the method according to paragraphs. 1-14, wherein said cell culture contains - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level. 19. Cell culture according to any one of paragraphs. 15-18, characterized in that it contains over 98% of MSCs that do not express CD11b, CD14, CD15, CD16, CD31, CD34, CD45, CD49f, CD102, CD104 and CD133 markers at a detectable level. 20. Pharmaceutical composition for the treatment of diabetes, containing at least 1 to 5 x 10 ⁶ cells of a cell culture obtained by the method of claim. 1, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 21. Pharmaceutical composition for the treatment of aplastic anemia, containing at least 1 to 5 x 10 ⁶ cells of a cell culture obtained by the method of claim 1, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 22. Pharmaceutical composition for the treatment of decompensated liver cirrhosis, containing at least 1 to 5 x 10 ⁶ cells of a cell culture obtained by the method of claim 1, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 23. Pharmaceutical composition for the induction of angiogenesis, containing at least 1 to 5 x 10 ⁶ cells of a cell culture obtained by the method of claim. 1, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 24. Pharmaceutical composition according to any one of paragraphs. 20-23, additionally containing a hydrogel or other biocompatible substance. 25. Cosmetic composition for the regeneration of skin or mucous cells, containing at least 1 to 5 x 10 ⁶ cells of a cell culture, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 26. Cosmetic composition for improving the appearance of the skin or mucous membrane, containing at least 1 to 5 x 10 ⁶ cell culture cells, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 27. Cosmetic composition for the treatment of damage or disorders of the skin, containing at least 1 to 5 x 10 ⁶ cells of a cell culture, including - over 60% of cells expressing CD106 at a detectable level, - over 98% of cells expressing CD151 at a detectable level, - over 98% of cells expressing nestin at a detectable level, - over 95% of cells expressing the markers CD73, CD90, CD105 and CD166 at a detectable level, and - less than 2% of cells that express CD45, CD34 and HLA-DR markers at a detectable level, and a biologically compatible substance. 28. System for the introduction of cell culture according to any one of paragraphs. 15-19 to the patient, which is a micro- or nanovesicles of biopolymers, lipids or nanoparticles, which include a cell culture according to any one of paragraphs. 15-19.

Images