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In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion
Andreea Ianus,1 George G. Holz,2,3 Neil D. Theise,1 and Mehboob A. Hussain3,4
1Department of Pathology,2 Department of Physiology and Neuroscience,3 Department of Pharmacology, and4 Department of Medicine, New York University School of Medicine, New York, New York, USA
Address correspondence to: Mehboob A. Hussain, Departments of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, MSB 424, New York, New York 10016, USA. Phone: (212) 263-0942; Fax: (212) 263-7133; E-mail: hussam02@popmail.med.nyu.edu.
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1Department of Pathology,2 Department of Physiology and Neuroscience,3 Department of Pharmacology, and4 Department of Medicine, New York University School of Medicine, New York, New York, USA
Address correspondence to: Mehboob A. Hussain, Departments of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, MSB 424, New York, New York 10016, USA. Phone: (212) 263-0942; Fax: (212) 263-7133; E-mail: hussam02@popmail.med.nyu.edu.
Find articles by Holz, G. in: JCI | PubMed | Google Scholar
1Department of Pathology,2 Department of Physiology and Neuroscience,3 Department of Pharmacology, and4 Department of Medicine, New York University School of Medicine, New York, New York, USA
Address correspondence to: Mehboob A. Hussain, Departments of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, MSB 424, New York, New York 10016, USA. Phone: (212) 263-0942; Fax: (212) 263-7133; E-mail: hussam02@popmail.med.nyu.edu.
Find articles by Theise, N. in: JCI | PubMed | Google Scholar
1Department of Pathology,2 Department of Physiology and Neuroscience,3 Department of Pharmacology, and4 Department of Medicine, New York University School of Medicine, New York, New York, USA
Address correspondence to: Mehboob A. Hussain, Departments of Medicine and Pharmacology, New York University School of Medicine, 550 First Avenue, MSB 424, New York, New York 10016, USA. Phone: (212) 263-0942; Fax: (212) 263-7133; E-mail: hussam02@popmail.med.nyu.edu.
Find articles by Hussain, M. in: JCI | PubMed | Google Scholar
Published March 15, 2003 - More info
J Clin Invest. 2003;111(6):843–850. https://doi.org/10.1172/JCI16502.
© 2003 The American Society for Clinical Investigation
Received: July 25, 2002; Accepted: February 11, 2003
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Methods
Murine model of bone marrow transplantation and experimental design. Animals were maintained under conditions approved by the institutional Animal Care and Use Committee at New York University School of Medicine. Transgenic mice expressing the phage CRE recombinase (CRE) under control of the rat insulin II promoter (INS2-CRE) (38) were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA; C57BL/6 background). Mice expressing enhanced green fluorescent protein (EGFP) at the ROSA26 locus (39) preceded by three floxed translation stop codons (ROSA-stoplox-EGFP; C57BL/6 background) (40) were obtained from G. Eberl and D. Littman (New York University School of Medicine). The stop codons are removed by activity of the CRE that recognizes the LoxP sequence and removes the chromosomal portion flanked by the LoxP sites (i.e., translation stop sequences), thus resulting in a permanent expression of EGFP (37). To generate INS2*EGFP mice, heterozygous INS2-CRE mice were crossed with hemizygous ROSA-stoplox-EGFP mice, and offspring were genotyped at the time of weaning. In INS2*EGFP mice, cells that activate the insulin promoter express CRE and become identifiable by their expression of EGFP. Genotyping was performed with the following primers according to standard protocols: EGFP (forward, 5′-GCGAGGGCGATGCCACCTACGGCA-3′; reverse, 5′-GGGTGTTCTGCTGGTAGTGGTCGG-3′; 450 bp), CRE (forward, 5′-TAAAGATATCTCACGTACTGACGG-3′; reverse, 5′-TCTCTGACCAGAGTCATCCTTAGC-3′; 250 bp).
Bone marrow from male donor mice was flushed under sterile conditions with HBSS from the medullary cavities of tibiae and femurs using a 25-gauge needle. Marrow cells were checked for viability with the trypan blue method and filtered (Nitex 03-50/31; Sefar America, Kansas City, Missouri, USA) as previously described (18). Recipients were irradiated female mice (1,050–1,100 cGy from a Gammacell 40 small-animal irradiator; Atomic Energy of Canada, Kanata, Ontario, Canada) and received approximately 106 unfractionated bone marrow–derived cells by tail vein injection. Recipient mice were kept in sterile cages with sterile chow and water for 2 weeks after irradiation and euthanized at 4–6 weeks after bone marrow transplantation for tissue harvesting. Because all animals had the same C57BL/6 background, no alloimmune or graft-versus-host response was expected (nor observed). We checked engraftment by determining the fraction of peripheral blood nucleated cells with a Y chromosome (two counts of 100 nucleated cells per animal).
Experimental bone marrow transplantation procedures are summarized in Figure 1. In one set of experiments, donor animals were male INS2*EGFP mice, and recipients were female wild-type mice (experiment 1). In a second set of experiments, donor animals were male INS2-CRE mice, and recipients were female ROSA-EGFP mice (experiment 2). Experiment 2 tested for EGFP expression in recipient cells secondary to any cell fusion with donor cells expressing CRE (see Discussion).
Figure 1
Mouse bone marrow transplantation protocol. Experiment 1: Bone marrow from male INS2*EGFP mice was injected into irradiated female wild-type mice. Experiment 2: Bone marrow from male INS2-CRE mice was injected into irradiated female ROSA-stoplox-EGFP mice.
Islet cell isolation. Islets were isolated using the collagenase method as previously described (41). Briefly, the pancreas was injected with 1 ml of collagenase P (2 mg/ml; Roche Diagnostics Corp., Indianapolis, Indiana, USA) and DNaseI (0.5 mg/ml; Roche Diagnostics Corp.) and incubated at 37°C in a total of 10 ml of digestion solution under constant shaking and intermittent vortexing. Islets were subsequently washed several times in HBSS with BSA (5 mg/ml) and hand-picked under a dissecting microscope. Islets were dispersed into single cells by suspension in trypsin EDTA (GIBCO BRL; Life Technologies Inc., Grand Island, New York, USA) and trituration through a siliconized Pasteur pipette. Cells were then cultured in RPMI 1640 (GIBCO BRL; Life Technologies Inc.), 5.5 mM glucose, 10% FCS in a humidified incubator (95% air, 5% CO2) at 37°C.
EGFP-positive cells were isolated from EGFP-negative cells by FACS (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA) or manual picking under a fluorescent microscope for further culture, RT-PCR (see below), or immunostaining and FISH. FACS data are given in Figure 4 and were generated on WinMDI 2.8 software (Joseph Trotter, Scripps Institute FACS core facility server, La Jolla, California, USA). Units of x and y axes are arbitrary but kept the same for all experiments. Parts a, e, and i of Figure 4 have linear axes and show forward and side scatter of cells. Parts b–d, f–h, and j–l have x and y axes in logarithmic scale and show green fluorescence intensity in the x axis (EGFP signal) and phycoerythrin (red) fluorescence intensity in the y axis (phycoerythrin signal).
Figure 4
Cell sorting and fluorescence analysis. (a–d) Peripheral blood nucleated cells; (e–h) bone marrow cells; (i–l) isolated and dispersed islet cells. (a, e, and i) Representational scatter plot of side (x axis) and forward (y axis) scatter of respective cells. (b–d, f–h, and j–l) Fluorescence scans (x axis, EGFP; y axis, phycoerythrin [red] filter) of respective cells. (b, f, and j) Cells from INS*EGFP donor mice (experiment 1). (c, g, and k) Cells from irradiated wild-type mice transplanted with bone marrow from INS*EGFP mice (experiment 1). (d, h, and l) Cells from irradiated ROSA-stoplox-EGFP mice transplanted with bone marrow from INS2-CRE mice (experiment 2). No fluorescence is detectable in donor or recipient peripheral blood nucleated cells or bone marrow cells. EGFP is detectable in islets of donor animals of experiment 1 and in the recipients of experiment 1. In b–d, f–h, and j–l, cells in the line equidistant from both axes show some autofluorescence, and cells in the lower right quadrant have EGFP signal. See Figure 1 for description of experiments 1 and 2.
Immunohistochemistry and FISH. Tissues were fixed in 4% paraformaldehyde for 2 hours at room temperature and subsequently in 30% sucrose overnight at 4°C before embedding in Tissue-Tek OCT compound (Sakura Finetek USA Inc., Torrance, California, USA) for sectioning (5–7 μm). A total of 200 pancreatic sections were analyzed. Isolated islet cells were fixed on microscope slides with 4% paraformaldehyde for 4 minutes at room temperature. Tissue sections and cells were treated with 100% methanol for 2 minutes at –20°C and then blocked 3% with normal donkey serum for 10 minutes at room temperature. Samples were then incubated at 4°C overnight with nonimmune serum, a guinea pig antiserum raised against insulin (Linco Research Inc., St. Charles, Missouri, USA) at a dilution of 1:1,000, rabbit anti–IPF-1 (1:500) (41), or rabbit anti-HNF3β (1:2,000) (provided by T. Jessel, Columbia University, New York, New York, USA; and A. Ruiz i Altaba, New York University School of Medicine). After rinsing with PBS, slides were blocked again with 3% donkey serum for 10 minutes at room temperature and then localized with donkey anti–guinea pig or donkey anti-rabbit with conjugated rhodamine X (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA) (1:500) for 30 minutes at room temperature. Slides were mounted in fluorescent mounting medium (Vector Laboratories Inc., Burlingame, California, USA) and stored in the dark. For frozen sections, a rabbit anti–green fluorescent protein IgG conjugated with Alexa Fluor 488 (Molecular Probes Inc., Eugene, Oregon, USA) at a dilution of 1:2,000 was used to detect the presence of EGFP-positive cells. After image capturing, the same samples were analyzed for the presence of mouse Y chromosome with FISH (Rainbow*FISH; Cambio Ltd., Cambridge, United Kingdom) according to the manufacturer’s recommendation. A digestion with proteinase K (10 μg/ml at 37°C, 5 minutes) (Sigma-Aldrich, St. Louis, Missouri, USA) was added at the beginning of the FISH protocol. Nuclei were stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) using fluorescent mounting medium containing DAPI at 1.5 μg/ml (Vector Laboratories Inc.).
Imaging was performed on a Zeiss Axioskop 2 fluorescent microscope (Carl Zeiss MicroImaging Inc., Thornwood, New York, USA) equipped with a cooled charge-coupled device digital camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan) and Improvision Open Lab software (Improvision Inc., Lexington, Massachusetts, USA) that allows pseudocoloring. Images were captured using the appropriate light absorption and emission filters supplied by the manufacturer of the microscope.
Reverse transcription and PCR. A subset of fluorescence-sorted islet cells were taken in culture for 24 hours and then hand-picked twice for positive EGFP fluorescence. One hundred of these cells were used for RT-PCR analysis. Total RNA from cells was purified using RNeasy with an RNase-free DNase digestion step (QIAGEN Inc., Valencia, California, USA). Reverse transcription was performed with Omniscript (QIAGEN Inc.), and PCR was performed using recombinant DNA polymerase (Takara Shuzo Co., Ltd., Shiga, Japan). Primers and PCR product size were as follows: insulin I (forward, 5′-TAGTGACCAGCTATAATCAGAG-3′; reverse, 5′-ACGCCAGGTCTGAAGGTCC-3′; 288 bp), insulin II (forward, 5′-CCCTGCTGGCCCTGCTCTT-3′; reverse, 5′-AGGTCTGAAGGTCACCTGCT-3′; 212 bp), IPF-1 (forward, 5′-TGTAGGCAGTACGGGTCCTC-3′; reverse, 5′-CCACCCCAGTTTACAAGCTC-3′; 325 bp), HNF3β (forward, 5′-ACCTGAGTCCGAGTCTGACC-3′; reverse, 5′-GGCACCTTGAGAAAGCAGTC-3′; 345 bp), HNF1α (forward, 5′-TCACAGACACCAACCTCAGC-3′; reverse, 5′-GAGGACACTGTGGGACTGGT-3′; 202 bp), HNF1β (forward, 5′-CCTAGGCTCCAACTTGGTCA-3′; reverse, 5′-TGTAGCGCACTCCTGACATC-3′; 203 bp), PAX6 (forward, 5′-AAGAGTGGCGACTCCAGAAGTTG-3′; reverse, 5′-ACCACACCTGTATCCTTGCTTCAGG-3′; 545 bp), CD45 (C57BL/6) (forward, 5′-AACTGGAACACTGCCTGCTT-3′; reverse, 5′-GACCACCTCACTGTCACGTTT-3′; 198 bp), oct-4 (forward, 5′-GGCGTTCTCTTTGGAAAGGTGTTC-3′; reverse, 5′-CTCGAACCACATCCTTCTCT-3′; 293 bp), cyclophilin (forward, 5′-CAGACGCCACTGTCGCTTT-3′; reverse, 5′-TGTCTTTGGAACTTTGTCTGCAA-3′; 132 bp). An oct-3/4 control plasmid was a generous gift from L. Dailey (New York University School of Medicine).
Functional characterization of islet cells: insulin secretion. After FACS sorting and/or manual picking, EGFP-positive islet cells (1,000 cells per microtiter well) were cultured for 24 hours before further analysis. Cells were then switched to medium containing either 5.5 mM or 11.1 mM glucose for an additional 10 hours. Some cells were also stimulated with the glucagon-like peptide-1 analog exendin-4 (Sigma-Aldrich; 10 nM) for the last 4 hours of incubation in 11.1 mM glucose. As control cells, isolated islet cells from INS2*EGFP mouse were taken for a parallel assay. Supernatant of islet cells was harvested, gently spun, and analyzed for insulin by ELISA (Ultra sensitive Insulin ELISA Kit; Crystal Chem Inc., Chicago, Illinois, USA).
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Results
We report on experiments 1 and 2 (Figure 1), conducted in parallel three times each with consistent results. A total of 48 animals were irradiated and injected with bone marrow cells. We observed an approximately 30% mortality rate in the first week after irradiation. Results of 21 animals in experiment 1 and 10 animals in experiment 2 are shown. Engraftment was found to be 70–90% when peripheral blood nucleated cells were examined for the presence of a Y chromosome.
On histological examination, we did not detect any changes suggestive of radiation injury. When wild-type female recipient animals were transplanted with bone marrow cells from male INS2*EGFP mice, EGFP-positive cells were detected within the islets of recipient mice (Figure 2) at 4–6 weeks after transplantation. On frozen sections (Figure 2) as well as in single-cell preparations (Figure 3, a–d), these EGFP-positive cells expressed insulin, as determined by immunohistochemistry. Furthermore, on FISH analysis, these cells revealed a Y chromosome within their nuclei, indicating that these cells were derived from the bone marrow of the male donor mouse (Figures 2 and 3). Bone marrow–derived cells from islets expressed not only insulin, but also transcription factors specific for β cells (IPF-1; Figure 3, e–h) and endoderm (HNF3β; Figure 3, i–l). In contrast, when ROSA-stoplox-EGFP female mice received bone marrow transplantation from INS2-CRE male donor mice (experiment 2), none of the cells within islets or the rest of the pancreas were fluorescent (Figure 2, b and d). Nevertheless, Y chromosome–positive cells were detectable within the islets, indicating that, in experiment 2, engraftment of bone marrow–derived cells did take place in the islet; no recombination events at the LoxP sites of recipient mice were detectable as assessed by absent immunostaining for EGFP (Figure 2d). In contrast, after we forced fusion in vitro (42) between INS2-CRE bone marrow cells and ROSA-stoplox-EGFP islet cells, we detected fluorescence (data not shown). These results indicate that in vivo cell fusion is unlikely to underlie apparent “transdifferentiation” of bone marrow–derived cells into a β cell phenotype.
Figure 2
Immunofluorescence and FISH for Y chromosome of pancreatic 5- to 7-μm frozen sections from recipient mice of experiments 1 and 2 (see Figure 1). (a and b) Bright-field phase images. (c and d) Composite overlay image of immunofluorescence for insulin (red) and EGFP (green), and FISH for Y chromosome (yellow) and DAPI staining of nuclei (blue). Y chromosome–positive cells (arrows) are present in the islet (i) and in the exocrine portions of the pancreas (e). EGFP is present only in insulin-positive cells in the islet. Scale bar, 10 μm; ×400. Insets in c and d are magnified areas of EGFP and insulin double-positive cells containing a Y chromosome.
Figure 3
Immunofluorescence and FISH of isolated, dispersed pancreatic islet cells from experiment 1 (see Figure 1). Images of identical fields are shown in the respective panels: (a–d) insulin, (e–h) IPF-1, and (i–l) HNF3β immunodetection. (a, e, and i)Bright-field phase; (b, f, and j) EGFP (note slight autofluorescence of control isolated islet cells); (c, g, and k) Immunostaining with rhodamine X–labeled secondary antibody for panels. (c) Insulin, (g) IPF-1, (k) HNF3β, and (dh, and l) FISH for Y chromosome (in yellow) and nucleus stain with DAPI (blue). Y chromosome is present only in EGFP-positive cells. Scale bar, 5 μm; ×630.
Fluorescence-activated cell analysis (104 cells counted per islet cell preparation) (Figure 4) and/or manual counting (300 cells counted)showed that, 4–6 weeks after bone marrow transplantation, the proportion of EGFP-expressing cells within the islet ranged from 1.7% to 3% of all cells within an islet, as compared with 80% to 85% in islets from control INS2*EGFP mice. Neither peripheral blood nucleated cells (Figure 4, a–d) nor bone marrow (Figure 4, e–h) from donor nor recipient mice revealed detectable EGFP in any of the experiments.
After sorting and culture for 24 hours, EGFP-positive cells from experiment 1 were further characterized by RT-PCR to express insulin I, insulin II, GLUT2, IPF-1, HNF1α, HNF1β, HNF3β, and PAX6, and to lack the common hematopoietic/leukocyte marker CD45 and the embryonic stem cell and bone marrow mesenchymal stem cell marker oct-3/4 (34) (Figure 5). This suggests that the bone marrow–derived cells that had engrafted the islets and that expressed EGFP did not express a non–β cell marker present in circulating nucleated cells or in bone marrow.
Figure 5
RT-PCR of isolated and FACS-sorted cells from experiment 1. (a) Peripheral blood nucleated cells (PBNCs) of recipient mice express cyclophilin (lane 1) as well as CD45 (lane 2), and EGFP-expressing cells derived from pancreatic islets express cyclophilin (lane 3), lack CD45 (lane 4), and express insulin (lane 5). (b) EGFP-expressing cells isolated from pancreatic islets do not express the stem cell marker oct-3/4. Lane 1: Positive control oct-3/4 plasmid. Bone marrow cells express cyclophilin (lane 2) and oct-3/4 (lane 3), and EGFP-expressing cells from pancreatic islets express cyclophilin (lane 4) but not oct-3/4 (lane 5). (c) EGFP-expressing cells isolated from pancreatic islets express cyclophilin (lane 1), insulin I (lane 2), IPF-1 (lane 4), and HNF3β (lane 5). Lane 3 is empty. (d) EGFP-expressing cells derived from pancreatic islets express cyclophilin (lane 1), insulin II (lane 2), insulin I (lane 3), GLUT2 (lane 4), HNF1α (lane 5), HNF1β (lane 6), and PAX6 (lane 7).
EGFP-positive cells responded to glucose as well as the glucagon-like peptide-1 synthetic analog exendin-4 (see Methods) with an insulin secretory response similar to that of control mouse islet cells that were analyzed in parallel (Figure 6). These results indicate that the cells studied have the machinery to sense glucose and the incretin hormone glucagon-like peptide-1 similarly to wild-type mouse β cells.
Figure 6
Insulin secretion of EGFP-positive cells isolated from islets is similar to insulin secretion of control islet cells. Insulin secretion after glucose or glucose and exendin-4 stimulation of isolated EGFP-positive cells from experiment 1 is shown as dark gray bars. Control cells were isolated from an INS*EGFP mouse and treated identically (light gray bars). One thousand dispersed islet cells were collected for EGFP expression by FACS and/or manual picking, cultured for 24 hours at 5.5 mM glucose or 11.1 mM glucose for 10 hours. Additional cells were incubated in 11.1 mM glucose for 10 hours and then with 10 nM exendin-4 for 4 more hours. Supernatant was collected for insulin measurements (ELISA). Results of three separate assays each performed in duplicate are shown as mean ± SD.
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