Abstract
Free full text
Increased Reprogramming of Human Fetal Hepatocytes Compared With Adult Hepatocytes in Feeder-Free Conditions
Abstract
Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar Syndrome, Type-1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with 6 factors, while fetal hepatocytes could be reprogrammed with 3 (OCT4, SOX2, NANOG) or 4 factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.
Introduction
Hepatocyte transplantation has been proposed as a cellular therapy for metabolic liver disease and acute liver failure. Primary hepatocyte transplantation has been used to partially correct patients with Crigler-Najjar, urea cycle, glycogen storage disorders and other metabolic liver diseases (1,3,5) and this therapy shows promise despite continuing issues with cell engraftment and proper immunosuppression. The availability of transplantable hepatocytes remains an ongoing problem.
Stem cell-derived hepatocytes from human embryonic stem cell (hESC) (22) and induced pluripotent stem cell (hiPSC) (20,23) technology has the potential to provide a nearly unlimited source of hepatocytes for basic research, or in vitro drug discovery if efficient protocols were developed to produce mature hepatocytes; however issues concerning tumorigenicity still limit the clinical application of hESCs or hiPSCs. Furthermore, hiPSC generation is limited by low reprogramming efficiency as well as long duration from induction to cell line establishment. hiPSCs have been derived from multiple cell types including fetal lung and neonatal foreskin fibroblasts, mesenchymal stem cells (14), mouse adult and fetal hepatocytes (7,9,18), amnion epithelial (13,24) and mesenchymal cells from the umbilical cord matrix or amnion (2) and even one report with human adult hepatocytes (11). Furthermore, hiPSCs have also been derived from fibroblasts from patients with different genetic disorders, including inherited metabolic disorders of the liver (16). These disease-specific cell lines potentially serve as an unlimited source of disease-specific cells if they could be differentiated back to a phenotype that faithfully recreates the disease. Theoretically, disease-specific cells could be genetically corrected and used for autologous cell transplantation to correct the phenotype. The idea of patient specific therapy has recently been put into question with a report that differentiated mouse iPSCs induce T-cell-dependent immune responses in syngeneic recipients (25).
Several groups have tried to increase reprogramming efficiencies by using all six transcription factors (10), by combination of transcription factors and chromatin modulation (4,17) or by addition of chemical compounds (12). However, if populations of cells are more easily reprogrammed, methods to increase reprogramming may not be necessary, and these cell populations could be useful for translational research and could also be good models for understanding the mechanisms of the reprogramming process.
Recently, an increased reprogramming capacity of mouse liver progenitor cells compared with differentiated liver cells was reported (7). There is evidence that mouse iPSCs retain epigenetic markings providing “memory” of the cell from which they were made (6) and that cell type of origin influences in vitro differentiation potential (15). A recent report demonstrated hepatic lineage stage-specific donor memory in mouse iPSCs, and interestingly, fetal hepatocyte-derived iPSCs demonstrated superior capacity for hepatic re-differentiation (9). Therefore, iPS from fetal human hepatocytes might be a particularly useful source of stem cell-derived hepatocytes.
There are no reports describing derivation of hiPSCs from human fetal hepatocytes or from hepatocytes from patients with an inborn error in metabolism. Here, we provide a method to generate hiPSCs from adult and fetal human hepatocytes under entirely feeder-free conditions and report that reprogramming of fetal hepatocytes was over 50-fold more efficient than adult hepatocytes and can be accomplished with only 3 reprogramming factors.
Materials and Methods
Cell Culture
The H1 hESC and all hiPSC lines were maintained on matrigel (BD Biosciences, San Jose, CA) and cultured in mTeSR™1 medium (Stem Cell Technologies, Vancouver, BC, Canada). Cells were passaged using 1 mg/ml dispase (Gibco, Carlsbad, CA) using conventional stem cell culture techniques. All cell lines used for data collection were passage 30 or under. The H1 hESC line was obtained from the WiCell Research Institute, Inc. Primary human hepatocytes were isolated using a collagenase perfusion method (8,19). Viable cell count was used for plating and plates were washed extensively to get rid of dead and unattached cells. Primary human hepatocytes were cultured in HMM (Lonza, Walkersville, MD) and supplemented with 1X penstrep (Invitrogen, Carlsbad, CA) and 10−7M insulin and dexamethasone (Lonza, Walkersville, MD). Primary human fetal hepatocytes were isolated by digesting the tissue in EMEM (Lonza, Walkersville, MD) containing 0.5 mg/ml of collagenase (Type XI, Sigma Chemical Co, St. Louis Mo, Cat. #C7657) on a lab quake shaker/rotisserie (Barnstead Intl. Dubuque, IA) for 40 minutes and otherwise processed as described for adult hepatocytes. Viable cell counts were used for plating and cultures were changed out with fresh media 2 hours after plating and washed extensively 18–24 hrs post plating to remove dead and unattached cells. Primary human fetal hepatocytes were cultured in DMEM (Lonza) supplemented with 10% FBS (PAA Laboratories, Pasching, Austria), 10−7M insulin, 1X penstrep, and 10 ng/ml EGF (BD Biosciences).
hiPSC Inductions
Primary human adult and fetal hepatocytes were added to matrigel-coated 6-well plates at 100,000 cells/well in their respective media and allowed to attach overnight. The following day transduction media was given to the cells that consisted of each cell type’s respective culture medium supplemented with 6 μg/ml polybrene and lentiviruses delivering up to six reprogramming factors OCT4, SOX2, KLF4, C-MYC, NANOG, and LIN28, (Thermo Fisher Scientific) each at a targeted MOI of up to 10 (Day 0). After 24 hours the cells were washed with DMEM/F12 (Thermo Fisher Scientific) 3X. Cells were cultured in their respective media and media was changed to mTeSR™1 on days 1–7. Media was refreshed every 2–3 days. Large hiPSC colonies appeared between days 16 and 21 and individual colonies were selected for expansion between days 25 and 35. Colony formation was determined by counting total number of individual hESC-like colonies formed in all wells. Reprogramming efficiency was determined by the following equation:
Table 1
Details of hiPSC induction experiments on adult and fetal hepatocytes. Information includes: donor number, age, sex, viability of cells after isolation and and the number of hiPSC lines identified,
| Donor # | Age Years | Sex | Viability (%) | hiPSC lines formed | Viruses used |
|---|---|---|---|---|---|
| HH1901 | 0.3 | M | 89.0 | No | OSKMNL |
| HH1904 | 0.6 | M | 84.0 | No | OSNLM |
| HH1806 | 0.9 | M | 70.5 | No | OSKMNL |
| HH1813 | 1 | M | 91.0 | No | OSKMNL |
| HH1892 | 1 | M | 88.0 | No | OSKMNL |
| HH1655 | 1.1 | F | 83.4 | AH-CN | OSKMNL |
| HH1815 | 1.1 | F | 90.0 | No | OSKMNL |
| HH1995 | 3 | F | 70.0 | No | OSNL |
| HH1764 | 10 | M | 76.0 | No | OSKMNL |
| HH1591 | 12 | F | 76.0 | AH1-2 | OSKMNL |
| HH1843 | 12 | F | 94.0 | No | OSKMNL |
| HH1996 | 14 | M | 70.0 | No | OSNL |
| HH1758 | 16 | F | 78.0 | No | OSKMNL |
| HH1705 | 39 | M | 96.0 | No | OSKMNL |
| HH1781f | 20 wks | Und | 95.0 | FH1 | OSN |
| HH1782f | 15 wks | M | 98.5 | No | OSNL |
| HH1791f | 12.7 wks | M | 91.0 | FH4-9 | OSNL |
| HH1794f | 18 wks | Und | 91.0 | No | OSNL |
| HH1795f | 21 wks | M | 91.0 | FH2-3 | OSNL |
| HH1902f | 22 wks | M | 95.0 | FH10 | OSKM |
| HH1902f | 22 wks | M | 95.0 | FH11-31 | OSNL |
| HH1986f | 24 wks | M | 96.0 | FH32-37 | OSNL |
Immunocytochemistry
hiPSCs were plated on matrigel-coated coverslips in 24-well plates, fixed using 4% PFA for 20 minutes at room temperature and permeabilized with 0.1% Triton X for 15 minutes for Oct4, Sox2, and Nanog at room temperature. Cells were not permeabilized for SSEA4. Cells were rehydrated with PBS and 0.5% BSA in PBS. Blocking was performed in 2% BSA for 45 minutes. Primary antibody was diluted in 0.5% BSA and incubated for 60 minutes. Secondary antibody was diluted in 0.5% BSA and incubated for 60 minutes. Nuclei were stained using Hoechst 33342 in PBS for 1 minute. Cells were mounted using gelvatol and refrigerated until images were captured on an Olympus Provis AX70 fluorescent microscope with Magnafire 2.1B software. The following primary antibodies were used: Rabbit anti-Oct3/4 (Santa Cruz sc-9081, Santa Cruz, CA) 1:250, Rabbit anti-Sox2 (Millipore AB5603) 1:300, Rabbit anti-Nanog (Abcam ab-80892, Cambridge, MA) 1:100, SSEA-4-Alexa Fluor 555 (BD Biosciences 560218) 1:100. The following secondary antibodies were used: Goat anti-rabbit Alex Fluor 488 (Invitrogen) 1:500 and Donkey anti-rabbit TR (Santa Cruz sc-2784) 1:250.
Teratoma Formation
hiPSCs were grown in 6-well plates till about 85% confluence. The entire 6-well plate was treated with 1 mg/ml dispase and cells were centrifuged at 200 × g for 5 minutes in a 50 mL conical tube. Cells were resuspended in 1 mL of mTeSR™1 and 1 mL of undiluted matrigel and equal volumes were transplanted subcutaneously between the scapulas of 3 NOD/SCID IL2γ-chain receptor knockout mice (dorsal injection). All animals were housed according to University of Pittsburgh Animal Husbandry protocols. Tumors were excised and fixed with 10% formalin overnight at 4°C. Tumor sections were processed and sections were made and stained for hematoxylin and eosin.
Gene Expression
RNA was isolated using TRIZOL® reagent (Life Technologies, Carlsbad CA) according to the manufacturer’s protocol. RNA integrity and purity were confirmed. DNase-I treated RNA of each sample were reverse-transcribed by PCR with RT-PCR reagents (Promega, Fitchburg, WI) according to manufacturer’s protocol. The resulting cDNA was analyzed by quantitative real-time PCR using specific TaqMan® assays and TaqMan® Gene Expression Master Mix (Applied Biosystems, Carlsbad, CA) according to the manufacturer’s protocol. Ct values were entered into the following equation to determine the arbitrary unit value: 1 × 109 × e(−0.6931 × Ct) (21) then normalized to cyclophilin A. All samples were run and analyzed on a ABI Prism 7000 Sequence Detection System. The following TaqMan® assays were used: Cyclophilin A (Hs99999904_m1), Oct4 (Hs03005111_g1), Sox2 (Hs01053049_s1), Nanog (Hs02287400_g1), Gdf3 (Hs00220998_m1), hTert (Hs00972656_m1), Dnmt3b (Hs00171876_m1), c-Myc (Hs00905030_m1), Klf4 (Hs00358836_m1), Baf155 (Hs00268265_m1), Brg1 (Hs00231324_m1).
Flow Cytometry
After a 3–5 minute exposure to accutase (Gibco), cells were counted and viability was determined. The cells were resuspended at a concentration of 107/ml in PBS enriched with human albumin and EGTA. We characterized the cells according their expression of nuclear and surface markers: NANOG, OCT3/4, SOX2, SSEA-3, SSEA-4, TRA1-60 and TRA 1-81 (all from BD Biosciences). The staining procedure was the same for both type of markers: a minimum number of 0.5 × 106 viable cells per tube were incubated for 45 minutes at 4°C with appropriate amount of monoclonal antibodies directly conjugated with six fluorochromes: fluorescein isothiocyanate (FITC), phycoerythrin (PE), cyanine chrome 5 (Cy5), allophycocyanine (APC), Alexa 466 (Al.466) and Alexa 667 (Al.667) (according to manufacturer dilutions). After staining, the samples were fixed with 2% paraformaldehyde for 10 min at room temperature. The evaluation of nuclear markers required a fixation-permeabilization procedure (Perm/Wash Buffer), as described in the manufacture protocol (BD Biosciences, San Jose, CA), before the staining procedure. Appropriate non-specific fluorescence-conjugated antibodies of identical isotype were used as negative controls in order to evaluate positive cells for each specific antibody used. Four color flow-cytometry acquisition was performed by dual laser FACSCalibur® equipped with CellQuest® software (BD Biosciences). To analyze and immunophenotype different cell subpopulations, at least 0.3 × 106 cells per tube were acquired. Sequential gating was implemented based on negative-control staining profiles. All samples were analyzed by FlowJo® software (Tree Star Inc., OR).
Transduction Efficiency
Primary human adult and fetal hepatocytes were plated on matrigel in 6-well plates at 100,000 cells/well in their respective media and allowed to attach overnight. The following day transduction media was given to the cells that consisted of each cell type’s respective culture medium supplemented with 6 μg/ml polybrene (Millipore, Billerica, MA) and GFP lentivirus (Thermo Fisher Scientific, Waltham, MA) at an MOI of 5, 10, 25, and 50 in triplicate wells (Day 0). After 24 hours the cells were washed with DMEM/F12 (Thermo Fisher Scientific) 3X and cells were given new cell culture media (Day 1). On Day 3 fluorescent images of the cells were taken using a Nikon Eclipse Ti inverted fluorescent microscope and Elements software (Nikon, Melville, NY). Ten random areas of each well from each MOI were taken for a total of 10 pictures per MOI. Transduction efficiency was determined by the following equation:
Toxicity Assays
Adult and fetal hepatocytes were isolated as described earlier. 10,000 cells/well were plated on 96-well plates coated with matrigel in their respective cell culture media and allowed to attach overnight. The next day each respective cell type’s media was supplemented with 6 μg/ml polybrene and a lentivirus delivering GFP (Thermo Fisher Scientific) at an MOI of 0–60. The next day the cells were washed and given their respective cell culture media. Cells for the 24 and 72 hour time points were analyzed for total double strand DNA using the Quanti-iT™ assay (Invitrogen), caspase activity by Caspase-Glo® 3/7 assay (Promega) and CellTiter-Glo® cell viability assay (Promega) according to manufacturer’s instructions.
Statistical Analysis
Reprogramming efficiency and gene expression analysis were analyzed using a two-tailed student’s t-test. The incidence of hESC-like colony formation was analyzed using a Fisher’s Exact Test where colony formation in an individual experiment was scored either as a yes or a no. All toxicity assays were analyzed using a two way ANOVA with a Bonferroni posttest. A p-value of <0.05 was determined as significant.
Use of Animal- and Human-derived Tissue
All animal experiments were performed in accordance with the University of Pittsburgh’s Institutional Animal Care and Use Committee. Furthermore all human tissue was obtained with informed consent and approved by the University of Pittsburgh’s Institutional Review Board.
Results
Donor characteristics for the hepatocyte cases used in these studies are presented in Table 1. For clarity, adult hepatocytes are defined here as any hepatocytes isolated from postnatal livers. Fetal hepatocytes were isolated from tissue ranging from 89 – 168 days of gestation (Table 1). Adult and fetal hepatocytes were freshly isolated and seeded on matrigel in their respective culture media and allowed to attach overnight The average viability according to trypan blue exclusion was 83% and 94% for adult and fetal cells, respectively. The cells were transduced with lentiviruses delivering the reprogramming factors for 24 hours and media was changed every 2 to 3 days until hESC-like colonies appeared (see Experimental Procedures). Large hESC-like colonies appeared between days 16 and 21 and individual colonies were selected for expansion between days 25 and 34 and were seeded onto new matrigel-coated plates in mTeSR™1 medium. Fully reprogrammed colonies were easily distinguishable by their similarity to hESC colonies (Figure 1). All selected colonies had high nuclei to cytoplasm ratio, well defined borders and were otherwise morphologically indistinguishable from hESCs (Figure 1). Three hiPSC lines were isolated from 2 different adult hepatocyte cases; one of which was from a 13 month old patient diagnosed with CN-1 syndrome and 2 lines were derived from a 12-year-old female organ donor. Attempts at reprogramming 12 other adult and pediatric cases failed to generate hiPSC colonies (n=18 total experiments with adult hepatocytes). These cases ranged from 4 months to 39 years. In contrast, 37 hiPSC lines were isolated from 5 different fetal hepatocytes cases. Importantly, one line, FH1, was reprogrammed with only 3 factors (OCT4, SOX2, and NANOG). Attempts at reprogramming 2 other fetal hepatocyte cases failed to generate hiPSC colonies (n=8 total experiments with fetal hepatocytes). Of the 40 lines generated, 8 were selected and were examined for characteristics of fully reprogrammed cells. Two adult (AH1 and AH-CN) and 6 fetal (FH1, FH2, FH12, FH14, FH15, FH29) hepatocyte hiPSC lines were further characterized for pluripotency for this report. All 8 of these hiPSC lines were characterized by morphology, gene expression, flow cytometry and ICC of pluripotency markers and teratoma formation. All other hiPSC lines established (both adult and fetal-derived) were expanded and cryopreserved and banked for later use. All of the characterized cell lines were carried out to at least passage 35 and maintained characteristic hESC-like morphology and a consistent proliferation rate.
Generation of hiPSCs from adult and fetal hepatocytes in entirely feeder-free conditions. Adult or fetal hepatocytes were given lentiviruses delivering the reprogramming factors in their respective culture media overnight (Day 0). The culture medium was changed to mTeSR™1 on Days 1–7. The pictures are representative images of hiPSC lines grown on matrigel in mTeSR™1. The hiPSC lines are morphologically indistinguishable from the H1 hESC line and grow in colonies with well defined borders and high nuclei to cytoplasm ratio. Original magnification of all pictures is X100. (A) Representative colony of fetal hepatocyte-derived hiPSC clone FH1. FH1 was derived using only OCT4, SOX2 and NANOG. (B) Representative colony of adult hepatocyte-derived hiPSC clone AH1. (C) Representative colony of adult hepatocyte-derived hiPSC clone made from cells of a patient diagnosed with CN-1. Clone AH-CN. (D) Representative colony of H1 hESC.
Quantitative RT-PCR was performed using human specific TaqMan® assays. The hESC line, H1, was used as a positive control and the IMR90 fibroblast line was used as a negative control. All hiPSC lines expressed OCT4, SOX2, NANOG, GDF3, hTERT and DNMT3B at levels comparable to H1 hESCs, although the latter 2 were the most variable between different hiPSC clones (Figure 2). Flow cytometry analysis demonstrated that the hiPSC lines analyzed expressed the pluripotency surface markers SSEA-3, SSEA-4, TRA1-60, and TRA1-81 and nuclear markers OCT4, SOX2, and NANOG at levels comparable to H1 hESCs (Figure 3). IMR90 fibroblasts were negative for all markers analyzed. The expression of TRA1-60 and 1-81 were the most variable between the different lines examined. Alkaline phosphatase activity, a property of pluripotent cells, was detected in adult and fetal-derived hiPSCs and H1 hESC; IMR90 fibroblasts were negative (data not shown). The data generated by flow cytometry was confirmed by the analysis of the expression and localization of expressed genes characteristic of reprogrammed cells by immunofluorescence. As with the flow data, the clones expressed nuclear OCT4, SOX2 and NANOG and surface marker SSEA4, and again IMR90 fibroblasts showed no specific reactivity (data not shown).
Gene expression analysis of pluripotency genes of hiPSC lines from adult and fetal hepatocytes. The H1 hESC line is used for a positive control and the IMR90 fibroblast line is used as a negative control. All expression level values are normalized to the cyclophilinA. All lines express the pluripotency markers OCT4, SOX2, NANOG, hTERT, GDF3 and DNMT3B.
Flow cytometry analysis of pluripotency marker expression of hiPSC lines from adult and fetal hepatocytes. The H1 hESC line is used for a positive control and the IMR90 fibroblast line is used for a negative control. All lines express the pluripotency markers SSEA3, SSEA4, TRA1-60, TRA1-81, OCT4, SOX2 and NANOG.
Teratoma formation is considered the definitive test of complete reprogramming to pluripotency. Each hiPSC line derived from either adult or fetal hepatocytes generated complex teratomas within 8–12 weeks in NOD/SCID IL2γ-chain receptor knockout mice. Representative pictures of structures resembling all three germ layer tissues, such as neural tissue and neural rosettes (ectoderm), chondroid tissue (mesoderm) and intestinal epithelium (endoderm) are shown in Figure 4.
Teratoma formation of selected clonally expanded hiPSC lines from both adult and fetal hepatocytes. The adult hepatocyte line depicted is AH1 and the fetal hepatocyte line depicted is FH1. The H1 hESC line is used for a positive control and the IMR90 fibroblast line is used for a negative control (data not shown). Cell lines were subcutaneously injected into the backs of NOD/SCID IL2γ-chain receptor knockout mice. All adult and fetal-derived hiPSC lines formed complex teratomas displaying cells from all three germ layers. FH1 and AH1 hiPSC lines display cell types ranging from neural tissue (ectoderm), chondroid tissue (mesoderm) and intestinal epithelium (endoderm).
Only 3 hESC-like colonies were identified from 18 experiments with 14 different donors when adult cells were used for attempted reprogramming. A total of 19,950,000 adult hepatocytes were exposed to reprogramming factors, and that resulted in 3 hiPSC colonies that were identified by their morphology and collected, expanded and cryopreserved. Vastly different results were obtained when reprogramming was attempted with fetal hepatocytes. A total of 80 hESC-like colonies were identified from 8 experiments with 7 different donors when fetal cells were used for attempted reprogramming. A total of 4,900,000 fetal hepatocytes were exposed to reprogramming factors. Of the 80 total hiPSC colonies generated from fetal hepatocytes, 37 hiPSC colonies with ES-like morphology were selected, expanded and cryopreserved. Reprogramming efficiency, defined as (number of hESC-like colonies formed/total number of cells exposed to the vectors) × 100, are 2.41 × 10−5 and 1.23 × 10−3, for adult and fetal hepatocytes, respectively (Figure 5A). This represents a highly significant, and over a 50-fold higher reprogramming efficiency of fetal hepatocytes as compared to adults (p=0.0015). Furthermore, 75% of the fetal hepatocyte cases yielded hESC-like colonies as compared to only 11% of the adult hepatocyte isolations (Figure 5B). The incidence, or likelihood, of hESC-like colony formation in any given experiment from adult vs. fetal hepatocytes is also highly significant (Fisher’s Exact Test with a p-value of 0.0028).
Fetal hepatocytes reprogram at over a 50-fold higher rate as compared to adult hepatocytes. A. Reprogramming efficiency, defined as (number of hESC-like colonies formed/total number of cells exposed to the vectors) × 100, was more than 50-fold higher for fetal hepatocytes as compared to adult hepatocytes. *p = 0.0015 versus adult hepatocytes and data are SEM of n=18 adult experiments and n=8 fetal experiments. (B) Fetal hepatocytes form hESC-like colonies at a higher incidence or likelihood than adult hepatocytes, Fisher’s Exact Test. *p = 0.0028 versus adult hepatocytes. Data are SEM of n=18 adult experiments and n=8 fetal experiments.
Fetal cells reprogrammed with a higher efficiency than the adult cells and so we examined the donor characteristics of the adult cases to determine if there was a consistent trend towards more efficient reprogramming in younger donors. As shown in Table 1, the cases where reprogramming was achieved were derived from 2 donors, one 12 years and the other 13 months of age. Reprogramming efficiency does not appear to be related to donor age in the adult cells, as the youngest “adult” donor, 4 months of age and 2 cases from 1-year-old donors also failed to reprogram. Many younger donors were examined and in total 11 of the 14 adult cases tested were from donors 12 years or under. Thus, from these 14 cases, there is no clear connection between donor age and reprogramming efficiency post birth. However, we did not specifically design the experiment to examine reprogramming efficiency in different age groups.
A trivial explanation for such large differences in reprogramming efficiency could be related to the infectivity of the cells with the lentiviruses. To determine if there were differences between adult and fetal cells with respect to transduction efficiency, a lentivirus carrying GFP was used in place of the reprogramming vectors at an MOI ranging from 0–50 and the percentage of GFP-positive cells was determined 72 hours after viral exposure (Figure 6A). The percentage of GFP-positive cells increased with increasing MOI in both fetal and adult cells. The data from adult cells was consistently higher than that observed with fetal cells, however there were no significant differences between adult and fetal cells at any MOI.
Fetal hepatocytes’ higher reprogramming efficiency is not due to transduction efficiency or toxicity to reprogramming factor viruses. (A) Adult and fetal hepatocytes were exposed to lentivirus delivering GFP at increasing MOI. There was no significant difference in transduction efficiency between adult and fetal hepatocytes. (B–D) The potential toxicity of lentiviral exposure was investigated at increasing MOIs and analyzed 24 hours after infection. Data was normalized to MOI 0. Toxicity was measured by caspase 3/7 activity (B), ATP content (C) and total double-strand DNA (D). Data are SEM of n=3 adult and fetal experiments. No statistical differences were seen between adult and fetal cells in the three assays.
Large differences in reprogramming efficiency could also result if reprogramming viruses were more toxic to adult cells. To test this, a lentivirus carrying GFP was used in place of the reprogramming vectors at MOI ranging from 0 to 60. Apoptosis levels were measured by caspase 3/7 activity and cell viability was measured by ATP content and total double strand DNA content 24 hours after viral exposure. All data was normalized to MOI 0. Adult and fetal hepatocytes showed no statistical differences between caspase 3/7 activity (Figure 6B), cell viability measured by ATP content (Figure 6C) or total double strand DNA levels (Figure 6D) at all tested MOI, however fetal cells trended towards higher caspase activity and lower amounts of ATP. Total double strand DNA levels were consistent between adult and fetal cells at all tested MOI. Similar data was obtained when toxicity was investigated 72 hours after viral exposure (data not shown).
It was previously shown in mouse that the increased reprogramming efficiency was associated with the endogenous expression of reprogramming factors (OCT4, SOX2, NANOG, KLF4 and C-MYC) and mediators of epigenetic changes during reprogramming (BAF-complex components BRG1 and BAF155) (7). Therefore quantitative RT-PCR was performed using human specific TaqMan® assays to determine the endogenous levels of expression of these genes in adult and fetal hepatocytes. Adult hepatocytes expressed higher levels of endogenous OCT4, SOX2, NANOG, and KLF4; while fetal hepatocytes showed higher expression of the two BAF factor complex members and C-MYC (Figure 7). Again, there were no statistical differences between expression of these genes in adult and fetal hepatocytes.
Endogenous gene expression analysis of reprogramming factors and BAF (Brg1/Brm associated factor)-complex members BAF155 and BRG1 in cultured adult or fetal hepatocytes. Expression level values are normalized to cyclophilinA. Adult and fetal hepatocytes expressed levels of all markers analyzed. Data are SEM of n=4 adult and n=3 fetal samples. No statistical differences were observed between adult and fetal cells.
Discussion
Here, we report the generation of 40 new hiPSC lines; derived from adult hepatocytes, and fetal hepatocytes. To the best of our knowledge this is the first report of generation of human fetal hepatocyte-derived hiPSCs, the generation of a metabolic disease specific hiPSC line from an adult hepatocyte (CN-1), and also the generation of hiPSC lines in entirely feeder-free conditions. Furthermore, in one attempt, derivation of fetal-derived hiPSCs was possible using only OCT4, SOX2, and NANOG. We did not use C-MYC, an oncogene associated with continued tumorigenicity of iPSC clones when they are differentiated from the pluripotent state.
Although initial experiments with adult hepatocytes were conducted with 4 reprogramming factors (n=4) and at 5 and 10 MOI for each factor, none of these experiments yielded reprogrammed colonies. Other investigators have reported that there is an enhanced efficiency of generating hiPSCs by a combination of six factors (10). Successful reprogramming of adult cells was finally accomplished when six factors were transduced. Fetal hepatocytes were found to reprogram over 50-fold more efficiently than adult hepatocytes even though only 3 or 4 reprogramming vectors were transduced. It was recently reported that increased reprogramming efficiency was obtained with a population of cells enriched for progenitor cells (7). Even though an enriched progenitor cell population was not used in our experiments, human fetal cells at the hepatoblast stage still show over a 50-fold higher reprogramming efficiency as compared to adult, in this feeder-free system.
Additional experiments demonstrated that the higher reprogramming efficiency of fetal cells was not due to a more efficient lentiviral infection of fetal hepatocytes than adult hepatocytes; as adult cells were actually transduced more efficiently than fetal cells (Figure 6A). Likewise, the differences in reprogramming efficiency were not due to the lentivirus being more toxic to the adult hepatocytes because at all tested MOI and time points the adult and fetal cells showed no statistical difference between levels of apoptosis, ATP content and total double strand DNA (Figure 6B–D). In fact, the trend was the viruses were more toxic to the fetal cells.
It was recently shown that an increased reprogramming efficiency of mouse liver progenitor cells, compared with differentiated liver cells, was associated with endogenous expression of reprogramming factors and BAF-complex members BAF155 and BRG1, which mediate epigenetic changes during reprogramming (7). The BAF-complex components achieve a euchromatic chromatin state and enhance reprogramming efficiencies by increasing the binding of reprogramming factors onto key pluripotency gene promoters (17). However when endogenous levels of expression of these markers in human adult and fetal hepatocytes were examined, adult hepatocytes trended to higher levels of expression of the reprogramming factors (Figure 7), although this was not statistically significant. Interestingly, fetal hepatocytes trended to higher expression levels of the BAF-complex members BAF155 and BRG1, although again this was not statistically significant (Figure 7). This suggests that the increased reprogramming capacity of human fetal hepatocytes in a feeder-free system is not due to higher levels of expression of the reprogramming factors or known mediators of reprogramming. Future experiments will be needed to determine if there is a molecular basis for increased reprogramming capability of human fetal hepatocytes because it is not conserved from the mouse models currently published. Increased reprogramming efficiency in fetal cells could simply be due to the observation that fetal human hepatocytes replicate robustly in culture even in the absence of added growth factors and are more likely to be simple diploid cells. In contrast, adult cells are mainly polyploid (4, 8 and 16 N) and proliferate poorly, in vitro, which together may make it more difficult to reprogram the adult hepatocytes.
Recent evidence suggests that at low passages numbers, mouse iPSCs may retain epigenetic “memory” of the donor cell from which they were made (6) and that cell type of origin influences in vitro differentiation potential (15). This has since been corroborated in liver when a group demonstrated hepatic lineage stage-specific donor memory to mouse iPSCs (9). More importantly these researchers showed that mouse fetal hepatocyte-derived iPSCs retained a superior capacity for hepatic re-differentiation, however this was lost after continuous passaging. Although, not studied here, future experiments will need to determine if human adult and fetal hepatocyte-derived hiPSCs differentiate efficiently to cells with mature hepatocyte characteristics and functions.
Fetal hepatocytes have some advantages for making new hiPSC lines as compared to adults. They can be generated in far fewer experiments, and at a lower expenditure of time and resources. With more clones to choose from, the best lines in terms of differentiation potential could be selected. Furthermore, fetal hepatocyte tissue serves as a potential source of tissue to generate disease specific hiPSCs; as many elective abortions are due to identification of genetic abnormalities. Moreover the fetal cells can be induced to pluripotency without the use of oncogenes such as C-MYC. Taken together, these results demonstrate that human adult and fetal hepatocytes can be reprogrammed to pluripotency using entirely feeder-free conditions and the efficiency of reprogramming fetal hepatocytes is more than 50-fold higher than that observed with adult cells. Although less frequent, fetal hepatocytes can be reprogrammed using only 3 factors, OCT4, SOX2 and NANOG. Human fetal and adult hepatocyte-derived hiPSCs may be a useful source for stem cell-derived hepatocytes that could be used for basic hepatic biology research, drug metabolism, and toxicology studies and possibly, as a cell source for hepatocyte transplants.
Acknowledgments
This work was funded in part by Pfizer, Inc., #N01-DK-7-0004/HHSN267200700004C and RC1-DK086135 (SCS) and National Institutes of Health grant T32EB001026 (MCH) and DK48794 (IJF).
Abbreviations
| BSA | bovine serum albumin |
| CN-1 | Crigler-Najjar Syndrome Type-1 |
| DMEM | Dulbecco’s modified eagle’s medium |
| Cyc | cyclophilin |
| DNMT3B | DNA (cytosine-5-)-methyltransferase 3 beta |
| EGF | epidermal growth factor |
| FBS | fetal bovine serum |
| GDF | growth differentiation factor |
| GFP | green fluorescent protein |
| hESC | human embryonic stem cell |
| hiPSC | human induced pluripotent stem cell |
| HMM | hepatocyte maintenance medium |
| hTERT | human telomerase reverse transcriptase |
| ICC | immunocytochemistry |
| IL2 | interleukin-2 |
| KLF4 | Kruppel-like factor 4 |
| MOI | multiplicity of infection |
| NOD/SCID | non obese diabetic severe combined immunodeficient |
| OCT4 | Octamer 4 |
| OTC | Ornithine Transcarbamylase |
| PBS | phosphate buffered saline |
| PFA | paraformaldehyde |
| PCR | polymerase chain reaction |
| SOX2 | SRY (sex determining region Y)-box 2 |
| SSEA | stage specific embryonic antigen |
| TRA | tumor rejection antigen |
References
Full text links
Read article at publisher's site: https://doi.org/10.3727/096368912x662453
Read article for free, from open access legal sources, via Unpaywall:
https://europepmc.org/articles/pmc3773298?pdf=render
Citations & impact
Impact metrics
Citations of article over time
Alternative metrics
Article citations
Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases.
Stem Cell Res Ther, 11(1):528, 09 Dec 2020
Cited by: 6 articles | PMID: 33298190 | PMCID: PMC7725121
Pluripotent stem cells with low differentiation potential contain incompletely reprogrammed DNA replication.
J Cell Biol, 219(9):e201909163, 01 Sep 2020
Cited by: 9 articles | PMID: 32673399 | PMCID: PMC7480103
Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.
PLoS One, 14(8):e0221762, 29 Aug 2019
Cited by: 11 articles | PMID: 31465481 | PMCID: PMC6715171
Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.
PLoS One, 14(4):e0215490, 25 Apr 2019
Cited by: 5 articles | PMID: 31022207 | PMCID: PMC6483185
Donor-Dependent and Other Nondefined Factors Have Greater Influence on the Hepatic Phenotype Than the Starting Cell Type in Induced Pluripotent Stem Cell Derived Hepatocyte-Like Cells.
Stem Cells Transl Med, 6(5):1321-1331, 01 May 2017
Cited by: 8 articles | PMID: 28456008 | PMCID: PMC5442714
Go to all (9) article citations
Similar Articles
To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector.
Stem Cell Res Ther, 10(1):185, 24 Jun 2019
Cited by: 16 articles | PMID: 31234949 | PMCID: PMC6591940
Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion.
J Biosci Bioeng, 120(2):210-217, 24 Jan 2015
Cited by: 6 articles | PMID: 25622768
Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.
Adv Exp Med Biol, 1376:151-180, 01 Jan 2022
Cited by: 4 articles | PMID: 34611861
Review
Residual expression of the reprogramming factors prevents differentiation of iPSC generated from human fibroblasts and cord blood CD34+ progenitors.
PLoS One, 7(4):e35824, 24 Apr 2012
Cited by: 40 articles | PMID: 22545141 | PMCID: PMC3335819
Funding
Funders who supported this work.
NIBIB NIH HHS (2)
Grant ID: T32EB001026
Grant ID: T32 EB001026
NIDDK NIH HHS (9)
Grant ID: RC1-DK086135
Grant ID: HHSN267200700004C
Grant ID: N01-DK-7-0004/HHSN267200700004C
Grant ID: DK48794
Grant ID: P01 DK096990
Grant ID: RC1 DK086135
Grant ID: HHSN267200700004G
Grant ID: R00 DK083556
Grant ID: R01 DK048794