Switching of mesodermal and endodermal properties in hTERT-modified and expanded fetal human pancreatic progenitor cells
© Cheng et al.; licensee BioMed Central Ltd. 2010
Received: 3 July 2009
Accepted: 15 March 2010
Published: 15 March 2010
The ability to expand organ-specific stem/progenitor cells is critical for translational applications, although uncertainties often arise in identifying the lineage of expanded cells. Therefore, superior insights into lineage maintenance mechanisms will be helpful for cell/gene therapy.
We studied epithelial cells isolated from fetal human pancreas to assess their proliferation potential, changes in lineage markers during culture, and capacity for generating insulin-expressing beta cells. Cells were isolated by immunomagnetic sorting for epithelial cell adhesion molecule (EpCAM), and characterized for islet-associated transcription factors, hormones, and ductal markers. Further studies were performed after modification of cells with the catalytic subunit of human telomerase reverse transcriptase (hTERT).
Fetal pancreatic progenitor cells efficiently formed primary cultures, although their replication capacity was limited. This was overcome by introduction and expression of hTERT with a retroviral vector, which greatly enhanced cellular replication in vitro. However, we found that during culture hTERT-modified pancreatic progenitor cells switched their phenotype with gain of additional mesodermal properties. This phenotypic switching was inhibited when a pancreas-duodenal homeobox (Pdx)-1 transgene was expressed in hTERT-modified cells with a lentiviral vector, along with inductive signaling through activin A and serum deprivation. This restored endocrine properties of hTERT-modified cells in vitro. Moreover, transplantation studies in immunodeficient mice verified the capacity of these cells for expressing insulin in vivo.
Limited replication capacity of pancreatic endocrine progenitor cells was overcome by the hTERT mechanism, which should facilitate further studies of such cells, although mechanisms regulating switches between meso-endodermal fates of expanded cells will need to be controlled for developing specific applications. The availability of hTERT-expanded fetal pancreatic endocrine progenitor cells will be helpful for studying and recapitulating stage-specific beta lineage advancement in pluripotent stem cells.
Cell therapy for diabetes mellitus will be advanced by the availability of additional donor cells. Islet cells may originate from pancreatic ductal or other epithelial cells, especially after damage in the adult pancreas [1, 2]. However, the replication potential of human pancreatic islet cells is limited, which generally restricts expansion of these cells under culture conditions . Although cell lines have been generated by oncogenetic transformation of islet cells [4, 5] tumorigenic cells will obviously be unsuitable for basic studies under various contexts, as well as for cell therapy. This problem of genetic transformation afflicts other sources of cells, for example, inducible pluripotential stem cells (iPS), which are teratogenic . Whereas reprogramming of cells through transcription factor modifications was recently found to be effective for transdifferentiating pancreatic exocrine cells to endocrine beta cells in vivo , generalization of this observation in developing treatments for diabetes mellitus requires much more work.
We consider fetal tissues to be of considerable interest as cell donors because these are particularly enriched in lineage-committed stem/progenitor cells. However, in comparison with pluripotent human embryonic stem cells (hESC) or iPS, the replication potential of fetal cells was limited [8, 9], possibly due to telomere shortening [3, 10]. Modification of fetal human liver cells with the catalytic subunit of human telomerase reverse transcriptase (hTERT) enhanced cell replication without loss of stem/progenitor cell properties and additional expression of pancreatic duodenal homeobox (Pdx)-1 in these cells induced regulated insulin expression [10, 11]. On the other hand, we recently determined that epithelial fetal human liver cells became altered under continuous culture, with development of a novel conjoint meso-endodermal phenotype under control of transcriptional regulation mechanisms . As nuclear transcription factors are of fundamental significance in directing embryonic development of the foregut endoderm, which originates both hepatic and pancreatic stem/progenitor cells , we considered that pancreatic fetal stem/progenitor cells may share this property of lineage switching.
Among lineage-specific mechanisms, some transcription factors are of particular significance in pancreatic lineage development, for example, Pdx1, Neurogenin 3 (NGN3), and others, while cytokine networks represent another level of endocrine regulation in stem cells, for example, activin A - a member of the transforming growth factor (TGF)-β superfamily - inducts beta cell differentiation in hESC [2, 12, 13].
To generate further insights into fetal human endocrine stem/progenitor cells, we isolated epithelial cells characterized by the display of epithelial cell adhesion molecule (EpCAM), and studied mechanisms of proliferation and differentiation. This led us to define phenotypic changes in cells during culture, including after increased cell replication with hTERT expression. We found that the initial epithelial/endodermal phenotype of fetal endocrine cells was altered with gain of mesenchymal/mesodermal phenotype, which was similar to changes in stem/progenitor cells isolated from fetal human livers, further emphasizing sharing of mechanisms in cells originating from the foregut endoderm . The ability to control the phenotype of these cells through additional manipulations offers potential ways to regulate cell differentiation for superior β cell functions and to understand mechanisms in stage-specific β lineage advancement in other types of stem/progenitor cells.
Materials and methods
Fetal tissues of 17 to 24 week gestation were from Human Fetal Tissue Repository of Albert Einstein College of Medicine (Bronx, NY, USA). A total of 20 fetal pancreata were used. Mature pancreatic islets were from Islet Distribution Program of Juvenile Diabetes Research Foundation New York, NY, USA. Procedures for fetal tissue collection, including informed consent in writing from donors, as well as this research were approved by the Committee on Clinical Investigations (Institutional Review Board) at Einstein.
Cell isolation and culture
Fetal pancreases were rinsed in Leffert's buffer (10 mM HEPES, 3 mM KCl, 130 mM NaCl, 1 mM NaH2PO4 and 10 mM glucose, pH 7.4) with 5 mM CaCl2, 100 U/ml DNase (Worthington Biochemical Corp., Lakewood, NJ, USA), and 0.03% collagenase P (Roche Applied Science, Indianapolis, IN, USA). Tissue was repeatedly passed through 5 ml syringe and gently agitated in this buffer for 20 to 30 minutes at 37°C. Dissociated cells were passed through 80 μm dacron (Sefar Filtration Inc. Depew, NY, USA) and washed twice in phosphate buffered saline (PBS, pH 7.2), 2 mM EDTA, 0.5% BSA (wash buffer) under 800 × g for five minutes at 4°C. After resuspending 5 × 107 cells in 0.3 ml wash buffer, FcR blocking reagent was added, and cells were incubated with 100 μl microbeads conjugated with antibody against human EpCAM (Miltenyi Biotec Inc, Bergisch Gladbach, Germany) for 30 minutes at 4°C. To verify cell separation, aliquots were incubated with FITC-conjugated anti-EpCAM (Miltenyi Biotec) for 10 minutes at room temperature and examined under epifluorescence. Remaining cells were resuspended in 1 ml PBS with pelleting of EpCAM-positive and -negative fractions in wash buffer under 300 × g for 10 minutes. Cell viability was determined by exclusion of 0.2% trypan blue dye.
For culture, 4 × 103 cells were plated in 35 mm dishes in Dulbecco's Minimal Eagle Medium (DMEM) (Life Technologies Inc., Rockville, MD, USA) with 2.5 mM glucose, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B, and 10% fetal bovine serum (FBS, Atlanta Biologicals Inc., Norcross, GA, USA) in 5% CO2 at 37°C. The medium was replaced twice weekly and near-confluent cells were subpassaged 1:3 with trypsin-EDTA for three minutes at 37°C. We analyzed primary cells after short-term (1 to 2 d) or long-term (10 to 14 d) culture, followed by serial passages until cell replication declined. Cells expressing hTERT were cultured for >50 passages. Changes in cell population doublings were determined during culture by manual counting of cell numbers. To induce insulin expression, cells were cultured in serum-free medium for seven days and with 4 nM human recombinant activin A (R&D Systems, Minneapolis, MN, USA) for another three days.
The vector encoding hTERT and puromycin selection marker was previously described . EpCAM-positive cells were transduced with 2 to 4 × 104 units/ml of hTERT retrovirus. Fresh DMEM was added after 24 h and cells were cultured to 70-80% confluency before adding 0.75 μM puromycin (Sigma Biochemical Co., St. Louis, MO, USA). A clone of EpCAM-positive cells transduced with hTERT was designated hTERT-FPC.
Pdx1 lentivirus vector (LV)
The pONY4-Pdx1 plasmid containing rat Pdx1 cDNA was provided by Dr. S. Efrat (Tel Aviv University, Tel Aviv, Israel). Pdx1 cDNA was excised by BamHI and SalI and 850 base pair fragment was subcloned into pCCLsinPPT.hPGK.IRES.GFP.Wpre plasmid to obtain transfer plasmid designated pCCLsinPPT.hPGK.Pdx1.IRES.GFP.Wpre. Pdx1-LV was produced by calcium phosphate transfection of 293T cells as described . pMDLg/pRRE was the HIV-derived packaging construct, pRSV-Rev construct expressed Rev regulatory protein and pMD2.G construct expressed VSV-G envelope protein. To obtain high-titer LV, 293T medium was concentrated under 50,000 × g for 140 minutes with tittering of LV in HeLa cells. To transduce cells, LV under 10 multiplicities of infection (MOI) was added to cells overnight at 37°C. Cell transduction was analyzed after 72 h with flow cytometry for green fluorescent protein (GFP).
3% Goat serum
AB3440, Guinea Pig anti-human insulin (Chemicon El Segundo, CA)
Rhodamine-conjugated anti-guinea pig IgG (AB7136, Abcam, Cambridge, MA)
3% Goat serum
AB932, Rabbit anti-Glucagon (Chemicon El Segundo, CA)
Goat anti-Rabbit IgG, Cy3-conjugated (AP132C, Chemicon)
3% Goat serum
ab14181, Rabbit anti-c-peptide (Abcam Inc, Cambridge, MA)
Goat anti-Rabbit IgG, Cy3-conjugated (AP132C, Chemicon)
5% Donkey serum
V212210, (United States Biologicals, Swampscott, MA)
Rhodamine-conjugated anti mouse IgG (#715-295-150, Jackson Immuno Research)
Cells were lysed for measuring insulin and c-peptide as previously described . Data were normalized to cell numbers.
Reverse-transcription polymerase chain reaction (RT-PCR)
Primer sequences (5'-3'): Forward and Reverse
Pancreatic duodenal homeobox1 gene (Pdx1)
Prohormone convertase (PC1/3)
Prohormone convertase (PC2)
Paired homeobox gene4 (PAX4)
Paired homeobox gene 6 (PAX6)
Glucose transporter2 (GLUT2)
NK homeobox protein 6.1 (NKX6 1)
NK homeobox protein 2.2 (NKX2 2)
GATA2 transcription factor
GATA6 transcription factor
Transforming growth factor alpha (TGFα)
Transforming growth factor beta1 (TGFβ1)
Transforming growth factor beta2 (TGFβ2)
Transforming growth factor beta1 receptor (TGFβ1R)
Transforming growth factor beta2 receptor (TGFβ2R)
Insulin-like growth factor receptor (IGFR)
α-Smooth muscle actin
Human telomerase reverse transcriptase (hTERT)
Glyceraldehyde phosphate dehydrogenase (GAPDH)
Cell transplantation studies
The Animal Care and Use Committee of Albert Einstein College of Medicine approved animal use in conformity with National Research Council's Guide for the Care and Use of Laboratory Animals (United States Public Health Service publication, revised 1996). All animals were maintained in the Institute of Animal Studies at Albert Einstein College of Medicine. We transplanted 2 × 106 hTERT-FPC modified by Pdx1 LV in NOD-CB17-prkdc-SCID mice (Jackson Labs., Bar Harbor, ME, USA) into portal vein as described previously . Animals were sacrificed in groups (n = 3 each) 24 hours and one, two or four weeks after transplantation to analyze cell engraftment and gene expression. To verify presence of transplanted cells in the liver, genomic DNA was isolated by Trizol Reagent. A primate-specific sequence from the Charcot-Marie-Tooth (CMT)-1A element was amplified by PCR and in situ hybridization was performed with a pancentromere probe to identify human cells as described . GFP immunostaining was performed as described . In some studies, tissues were additionally stained for insulin as described above. After washing with PBS, tissues were counterstained with DAPI (4',6-diamidino-2-phenylindole)-antifade (Molecular Probes, Invitrogen Life Technologies Corporation, Carlsbad, CA, USA) and examined under epifluorescence.
Data are shown as means ± SD. Significances were examined by t-test. P values < 0.05 were considered significant.
Characterization of pancreatic epithelial cells
Proliferation of EpCAM-positive cells after telomerase reconstitution
Restoration of endocrine phenotype in hTERT-FPC
Endodermal-mesenchymal phenotype conversions
Transplantation of hTERT-FPC cells in NOD/SCID mice
We successfully isolated epithelial progenitor cells from the fetal human pancreas and isolated cells were greatly expanded after genetic modification with hTERT in vitro. These cells exhibited endocrine progenitor phenotype with multiple pancreatic hormones, although this phenotype was altered in culture conditions with acquisition of additional mesenchymal/mesodermal properties. As this phenotype change occurred through spontaneous processes, we believe the availability of hTERT-modified pancreatic cells will offer additional substrates for dissecting regulatory mechanisms in beta cell differentiation. In particular, our findings should be of interest in determining whether other stem/progenitor cells will transition through similar fetal stages during generation of β or beta-like cells.
During development, pancreatic progenitor cells arise from the primitive foregut endoderm, with cell-specification requiring Pdx1 and other transcription factors, for example, HNF homeobox B, NGN3, pancreas-specific transcription factor 1a, GATA4, and HNF6 . We were able to isolate pancreatic epithelial progenitor cells characterized by the epithelial-specific adhesion molecule, EpCAM, which is a feature of endodermal stem/progenitor cells [8, 9], although these cells demonstrated limited proliferation capacity in culture conditions. The replication potential of fetal human liver stem/progenitor cells expressing EpCAM was similarly limited and was accompanied by telomere shortening, which could be circumvented by hTERT expression . In the same way, modification of pancreatic progenitor cells with hTERT greatly expanded their capacity to proliferate. The ability of other somatic cells, and even iPS cells, to assume greater proliferation capacity after modification with hTERT has been established [6, 10]. This role of hTERT should be noteworthy because of the absence of tumorigenicity in hTERT-modified cells [10, 20], as shown previously, and again in our cell transplantation studies here, where transplanted cells did not generate foci of proliferating cells and no tumors were observed over at least four weeks.
We do not propose that genetically-modified cells with hTERT expression will be appropriate for immediate clinical applications, as more information will be needed in regards to their biological properties, regulation of differentiation through nonintegrating vectors in case of gene therapy-type approaches or of alternative small molecule approaches for this purpose, as recently identified during β lineage derivation in hESC . The availability of fetal cells described here will permit comparative studies of differentiation mechanisms in pluripotent stem cells versus stem/progenitor cells with specific lineage commitment. Our preliminary studies indicated that (-)-indolactam V, which was effective for inducing beta cell maturation in hESC-derived cells , failed to enhance beta cell phenotype in hTERT-FPC, suggesting fundamental differences in regulation of beta cell differentiation in various types of stem/progenitor cells.
The loss of insulin expression in cultured FPC was in agreement with previous studies, for example, downregulation of hormone expression was a feature of immortalized human pancreatic cell lines, as well as of growth-stimulated primary human pancreatic cells [22, 23]. Whether loss of gene expression was due to transcriptional downregulation, independent of cell differentiation, or switching of progenitor cells to more primitive states, were both possibilities. In our studies, EpCAM-positive fetal epithelial cells displayed properties of islet progenitor cells with expression of multiple hormones and relevant transcription factors in early cell culture conditions. We found that loss of insulin expression was coupled with simultaneous loss of Pdx1 expression in hTERT-FPC. Subsequently, we found that although this initial epithelial/endodermal phenotype was maintained in cells, additional properties typically encountered in mesenchymal/mesodermal cells, for example, vimentin expression, were displayed. This was strongly reminiscent of changes in EpCAM-positive fetal human liver stem/progenitor cells, where downregulation of the epithelial/endodermal phenotype was due to the onset of conjoint meso-endoderm (epithelial plus mesenchymal) phenotype under culture conditions . Also, these fetal human liver cells regained expression of epithelial genes after culture under serum-free conditions, which strengthened sharing of mechanisms observed in fetal pancreatic cells here.
Therefore, our findings of increased insulin expression in hTERT-FPC following culture under serum-free conditions with activin A were in agreement with the role of pathway-specific soluble signals in cell differentiation. The role of activin A in enhancing insulin expression in hTERT-FPC resembled the role of inductive signaling in generating β-like cells from hESC , and of extrinsic signaling in inducing hepatic properties in fetal stem/progenitor cells . These shared mechanisms will offer new opportunities for exploring genetic and epigenetic processes in pancreatic lineage regulation, including induction of the beta cell phenotype. The availability of hTERT-FPC and hTERT-FH-B cells derived from fetal liver and pancreas, respectively, should provide important opportunities for comparing hESC- or iPS-derived cells in understanding transitions of embryonic-to-fetal and fetal-to-adult stages in the β cell lineage. For instance, it should be relevant to determine whether candidate approaches capable of advancing β cell maturation in hESC-derived cells, could be equally effective in hTERT-FPC or hTERT-FH-B cells.
As hTERT-FPC were capable of expressing insulin in vivo, this was similar to the expression of hepatic functions in fetal human liver epithelial cells following transplantation in xenotolerant mice . In other studies, transplantation of hTERT-FH-B fetal liver cells modified by Pdx1, was successful in correcting hyperglycemia in mice [11, 24]. Here, our cell transplantation studies were limited to establishing differentiation of hTERT-FPC in the liver. Whether these cells could correct hyperglycemia in animals will require detailed studies in the future, particularly after further differentiation and maturation of cells along the β lineage.
Insights into lineage-switching mechanisms during expansion of pancreatic stem/progenitor cells will be critical for understanding the fundamental nature of expanded cells. The availability of hTERT-FPC should facilitate efforts to identify effective mechanisms to maintain stem/progenitor cells under suitable states for cell expansion without extinction of differentiation capacity. This will be useful for translational applications, including cell therapy in type-1 diabetes mellitus.
bovine serum albumin
complementary deoxyribonucleic acid
Dulbecco's Minimal Eagle Medium
dipeptidyl peptidase IV
epithelial cell adhesion molecule
fetal bovine serum
GATA family of transcription factors
green fluorescent protein
human embryonic stem cells
hepatic nuclear factor
human telomerase reverse transcriptase
human telomerase reverse transcriptase containing fetal pancreatic cells
insulin-like growth factor-1 receptor
inducible pluripotential stem cells
: potassium chloride
sodium dihydrogen phosphate
NK homeobox protein
natural onset diabetes-severe combined immunodeficiency
paired homeobox gene
phosphate buffered saline
third generation lentivirus plasmid
pancreatic duodenal homeobox gene
plasmid construct expressing vesicular stomatitis virus G envelope protein
plasmid containing human immunodeficiency virus-derived packaging construct
plasmid construct expressing Rev regulatory protein
reverse-transcription polymerase chain reaction
transforming growth factor.
This work was supported in part by NIH grants P01 DK52956, P20 GM075037, R01 DK46952, R01 DK071111, P30 DK41296 and P30 CA13330. Mark Zern, MD, University of California, Davis, provided hTERT retrovirus and E. Vigna contributed to the lentiviral vector.
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