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TISSUE-SPECIFIC STEM CELLS

Human Islet-Derived Precursor Cells Are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone-Expressing Cells In Vivo

Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

Key Words. Islet precursor cells • Mesenchymal stromal cells • Insulin • Cell replacement therapy • Diabetes

Correspondence: Marvin C. Gershengorn, M.D., Clinical Endocrinology Branch, Building 50, Room 4133, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. Telephone: (301) 496-4128; Fax: (301) 496-9943; e-mail: marving{at}intra.niddk.nih.gov

Received on April 30, 2007; accepted for publication on September 5, 2007.

First published online in STEM CELLS EXPRESS  September 27, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin-producing cells. Previously, we described proliferative populations of human islet-derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000-fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C-peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105⁺/CD73⁺/CD90⁺ cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas-derived MSC that are capable of differentiating into hormone-expressing cells. Their ability to mature into functional insulin-secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet-like cells for replacement therapy in type 1 diabetes.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Cell replacement therapy for type 1 diabetes offers improved glucose homeostasis, but transplantation of cadaveric islets, although effective in patients for a restricted period of time [1, 2], is limited by the supply of donor pancreases. Undifferentiated islet or β-cell precursors are promising because they can expand exponentially prior to differentiation [3–9] and thereby potentially produce a large supply of functional insulin-producing cells. We generated proliferative populations of human islet-derived precursor cells (hIPCs) in vitro from preparations of adult human islets and showed that hIPCs differentiate in vitro into insulin-expressing islet-like cell aggregates (ICAs) and, preliminarily, that ICAs transplanted under the kidney capsule of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice secreted insulin [10]. In that study, we presented evidence consistent with the idea that hIPCs were derived from insulin-expressing cells. More recently, we [11] and others [12–14] found no evidence by lineage analysis that mouse islet-derived precursor cells (IPCs) are derived from beta cells. If IPCs from mouse and human islets have similar origins, it is likely that hIPCs are not derived from human beta cells. The origin of hIPCs has, however, not yet been determined.

In this report, we further characterize hIPCs. We use hIPCs expanded more than 1,000-fold to generate immature epithelial cell clusters (ECCs) after 4 days of differentiation in vitro in the absence of added growth/differentiation factors. After transplant into mice, some ECC preparations differentiate and mature into functional ICAs that secrete human C-peptide in response to glucose and express transcripts for insulin, glucagon, and somatostatin at approximately 1% of the levels in adult human islets. Moreover, we show that hIPCs exhibit many characteristics of mesenchymal stromal cells [15], including adherence to plastic tissue culture dishes, expression of specific cell-surface markers including THY1 T-cell antigen (CD90), endoglin (CD105), and 5'-nucleotidase (CD73), sustained high proliferation, and capacity to be induced to differentiate into adipocytes, chondrocytes, and osteocytes. Indeed, we find hIPC-like CD105⁺/73⁺/90⁺ cells in fresh islets that have not been incubated in vitro. Most importantly, hIPCs provide an easily available and abundant supply of mesenchymal stromal cells (MSCs) with capacity to differentiate into hormone-expressing islet-like cells.

	MATERIALS AND METHODS

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hIPC Culture
hIPCs were derived from fresh human islets as described previously [10] and maintained in 150-mm tissue culture dishes in growth medium. Growth medium was CMRL-1066 (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 10% Fetal Bovine Serum, Prime (Biosource, Rockville, MD, http://www.invitrogen.com), 2 mM L-glutamine, and penicillin/streptomycin. Cells were passaged every 3–4 days at 80%–90% confluence. Briefly, growth medium was removed, and 10 ml of Trypsin EDTA (Cellgro; Mediatech, Manassas, VA, http://www.cellgro.com) was added per dish. After 2–5 minutes at 37°C, an equal volume of growth medium was added, and cells were collected by centrifugation. Cells were resuspended in fresh growth medium, counted using a Vi-Cell XR analyzer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com), and seeded in new dishes at 1.5 x 10⁴ cells per cm². Over the initial 20–25 passages, cell number approximately doubled with each passage.

For the formation of ECCs, cells were monodispersed with trypsin, collected by centrifugation, and washed twice with differentiation medium. Differentiation medium was CMRL-1066 supplemented with 2 mM L-glutamine, 1% (wt/vol) bovine serum albumin (BSA) Fraction V Fatty Acid Free (MP Biomedicals, Irvine, CA, http://www.mpbio.com), and 1x insulin-transferrin-selenium-A (100x; Gibco). Of note, we found that cells cluster more readily in fatty acid-free BSA than in BSA not treated to remove fatty acids. In addition, monodispersion of mesenchymal cells with trypsin is essential for differentiation since adherent cells refed with differentiation medium do not migrate, do not form clusters, and do not differentiate efficiently. After washing, cells were counted, diluted with differentiation medium, and transferred to 6-well non-tissue culture-treated plates (catalog number 351146; Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.co) at 3.3 x 10⁵ cells per well. A portion of these day 0 cells were collected by centrifugation and used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis. One day after transfer to differentiation medium, developing ECCs were collected by centrifugation (50g, 90 seconds), resuspended in fresh differentiation medium, and redistributed into their original 6-well plates. On day 4, approximately 12–16 hours prior to transplantation, ECCs from three 6-well plates were pooled using gentle pipetting to recover any minimally adherent clusters, collected by centrifugation (50g, 1 minute), resuspended in differentiation medium, and transferred to a 100-mm untreated Petri dish (Becton, Dickinson and Company).

Transplantation
ECCs generated from mesenchymal hIPCs that had been expanded by 10 or more doublings were used for transplantation. This means that the cells used for implantation have proliferated to generate at least 1,000-fold (2¹⁰) more cells than were initially present in the islets used to establish the hIPC culture. For each mouse to be transplanted, 1,000–1,500 day-4 ECCs, which typically ranged from 50–200 µm, were collected by centrifugation at 100g for 5 minutes, and 100 pg of vascular endothelial growth factor (V7259; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was added to the pellet. Generally, a total of approximately 2 x 10⁶ cells were transplanted into each mouse. ECCs were maintained on ice until transplantation. Male NOD/SCID mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) aged 8–12 weeks were sedated using isoflurane. Prior to surgery, 15 µl of tail-vein blood was collected from each mouse, added to the pellet designated for that mouse, and mixed to ensure that all cell clusters were engulfed in the resulting clot. The left kidney was exposed, and the blood clot containing hIPC ECCs was placed into a small pocket created under the renal capsule. Animals were housed individually after surgery and allowed food and water ad libitum.

At various times after transplantation, mice were fasted overnight and glucose was injected intraperitoneally at 2 mg/g body weight. Blood samples were obtained from tail vein before and 30 minutes after glucose injection. For terminal bleeds, animals were sedated using isoflurane, and blood samples were taken from the ventricle. Serum samples were assayed using a human C-peptide enzyme-linked immunosorbent assay kit (Millipore, Billerica, MA, http://www.millipore.com) or a mouse C-peptide radioimmunoassay (Millipore).

Quantitative Polymerase Chain Reaction
Total RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany, http://www.qiagen.com). First strand cDNA was prepared using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Polymerase chain reaction (PCR) was performed in 25-µl reactions in 96-well plates using cDNA prepared from 100 ng or less of total RNA and Universal PCR Master Mix (Applied Biosystems). Primers and probes were Assay-on-Demand (Applied Biosystems). We have confirmed linear amplification of insulin and glucagon transcripts by quantitative (q)RT-PCR on samples containing varying proportions of total RNA from human islets and HeLa cells. This linear relationship continued until the fluorescence cycle threshold (Ct) values were 38, and we therefore consider Ct values greater than 38 to be undetectable. Results for mRNA levels in Figure 4 and supplemental online Figure 2 are presented as a percentage of the values in fresh human islets after normalizing for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Thus, a normalized insulin Ct value of 28 in a recovered graft would be reported as 0.00153% relative to an average insulin Ct value of 12 in fresh islets (2^12–28 = 2^–16 = 0.0000153 = 0.00153%). We determined that the human GAPDH primer-probe set did not detect mouse GAPDH. qRT-PCR results from RNA isolated from ICA grafts were normalized to human GAPDH levels to correct for any contaminating mouse RNA in the recovered sample.

Antibodies and Immunostaining
Rabbit antibodies to human C-peptide (Millipore) and mouse antibodies to human glucagon (Sigma) were used at 1:100 dilution in blocking buffer (4% donkey serum in Dulbecco's phosphate-buffered saline [DPBS]). Alexa-Fluor 488, 546, and 633 F(ab')2 secondary antibodies (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were used at 1:200 dilution. Transplant-containing kidneys from euthanized mice were fixed in 4% paraformaldehyde and embedded in paraffin, and 10-µm sections were prepared. Paraffin-embedded sections were deparaffinized following standard procedures. For antigen retrieval, sections were incubated in citrate buffer (10 mM sodium citrate, pH 6.0) for 20 minutes at 95°C. After incubation in blocking buffer for 30 minutes at room temperature, sections were incubated with primary antibodies for 1 hour at 37°C, washed three times with DPBS, and incubated with secondary antibodies at 37°C for 1 hour. Slides were then washed extensively with DPBS and mounted in Prolong Gold antifade reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Confocal images were captured with a Zeiss LSM 510 Meta NLO laser scanning inverted microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Fluorophore-labeled antibodies for fluorescence-activated cell sorting (FACS) analysis were against human CD73 (phycoerythrin [PE]), CD90 (fluorescein isothiocyanate [FITC]), CD19 (allophycocyanin [APC]), CD34 (PE), CD14 (Alexa 488) (all from BD Biosciences, San Diego, http://www.bdbiosciences.com), and CD105 (APC) (eBioscience Inc., San Diego, http://www.ebioscience.com). Antibodies were used at the dilution recommended by the manufacturer.

Differentiation of hIPCs to Adipocytes, Chondrocytes, and Osteocytes
To induce adipogenesis, chondrogenesis, and osteogenesis, hIPCs were cultured in the appropriate induction media according to the manufacturer's protocols (Cambrex, Walkersville, MD, http://www.cambrex.com). The differentiated phenotype was documented using oil red O staining for adipocytes, von Kossa staining for osteocytes, and Alcian Blue staining for chondrocytes.

Analysis of Cell-Surface Markers and Cell Sorting
Human islets were treated with 0.05% trypsin/EDTA solution for approximately 5 minutes at 37°C until most cells were monodispersed. Monolayer cultures of hIPCs or human bone marrow-derived MSCs were monodispersed using trypsin as described above. Monodispersed cells were washed once with serum-containing medium, collected by centrifugation, and washed once with phosphate-buffered saline (PBS) at 4°C. Cells were resuspended in cold PBS containing 10% donkey serum at a concentration of 10⁷ cells per milliliter and transferred to round-bottom 12 x 75-mm polystyrene tubes. Cells were maintained at 4°C or on ice for all subsequent procedures. After 20 minutes, primary or isotype control fluorophore-labeled antibodies were added, and incubation continued for a minimum of 40 minutes. Immediately prior to FACS analysis, samples were diluted 10-fold with cold PBS. Cells were analyzed for fluorescence using a FACSCalibur (BD Biosciences). Cells were sorted using a FACSVantage SE equipped with the DiVa option. For RNA transcript analyses, cells were first sorted using the preset purity sort precision mode into CD105⁺/CD73⁺/CD90⁺ and CD105^–/CD73^–/CD90^– populations. Each population was then sorted again using the identical sorting gates and settings, and 10, 20, 40, 80, 160, 320, or 640 positive or negative cells were delivered directly into collection tubes containing 200 µl of cell lysis buffer RLT (Qiagen) containing β-mercaptoethanol. RNA was prepared using an RNeasy Micro Kit (Qiagen). Carrier RNA was added to each sample as recommended by the manufacturer. cDNA was prepared in a 20-µl reaction using the Sensiscript RT Kit (Qiagen). Real-time PCR was performed as described above using a 25-µl final volume containing 2 µl of the cDNA reaction product.

The issue of possible contamination of sorted CD105⁺/CD73⁺/CD90⁺ cells with insulin mRNA released from damaged beta cells in the islet preparations was addressed in two ways. First, triple positive cells from the initial sort were collected by centrifugation, and the supernatant was filtered through a 0.22-micron filter to assure removal of all cells. RNA and cDNA were prepared from aliquots of this supernatant as described above. Insulin transcript was not detected by quantitative PCR in 20-µl aliquots of this supernatant, which represents a much larger volume than the approximately 1 µl delivered when sorting 80 cells under our experimental conditions. This indicates that the insulin transcript present in CD105⁺/CD73⁺/CD90⁺ cells from human islets is not due to contamination by soluble insulin mRNA released from insulin-positive cells in the islet preparation. Second, human bone marrow-derived MSCs were harvested from monolayer cultures using the same solution of trypsin/EDTA that had been used immediately prior to monodisperse fresh human islets. These MSCs were then added to a portion of the monodispersed islet cells in at least a 200-fold excess to the expected number of islet-derived triple-positive cells. After labeling with antibodies to the three CD markers as described, CD105⁺/CD73⁺/CD90⁺ were sorted and resorted into collection tubes, RNA and cDNA were prepared, and insulin transcript was found to be undetectable by quantitative PCR in samples containing as many as 1,280 cells. This result suggests that the insulin transcript found in CD105⁺/CD73⁺/CD90⁺ cells from freshly isolated islets is not due to nonspecific adsorption or uptake of insulin transcript that may have been released from islet beta cells during islet dispersion and processing.

Statistical Analysis
Statistical differences were calculated by two-tailed Student's t test at p < .05.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We began our characterization of hIPCs using established preparations that do not express insulin mRNA after multiple passages in vitro but that can be induced to differentiate into insulin-expressing cells in vitro [10]. To further characterize the differentiation potential of established cultures of hIPCs, we studied clusters derived from hIPCs after transplantation into mice. Our protocol for deriving expanded populations of hIPCs and differentiating these cells into ICAs in mice is summarized schematically in Figure 1. hIPCs that had been expanded at least 10 population doublings and in which insulin transcript was not detectable were harvested and replated in serum-free medium to induce differentiation. Time-lapse studies illustrate that replated hIPCs adhere and migrate as mesenchymal cells within the first few hours of exposure to differentiation medium, forming distinct ECCs within 15–18 hours (supplemental online Fig. 1). Replating was necessary because cells that remained attached to the tissue culture dish did not differentiate as efficiently as cells that migrated into clusters. Cells within ECCs were rounded and exhibited fold increases in expression of transcripts for the characteristic epithelial junctional proteins claudins 3 and 4 of 9.2 ± 2.0 and 6.5 ± 2.4 (n = 4), respectively, when compared with proliferative hIPCs. This morphological change from adherent monolayer cells to rounded, clustered cells and upregulation of epithelial transcripts suggested that these serum-free conditions had suppressed cell growth and initiated epithelial differentiation. However, ECCs at day 4 did not express C-peptide or glucagon peptide (data not shown) and insulin mRNA was undetectable using a highly sensitive (q)RT-PCR assay, suggesting that the level of insulin transcript was at least 10 million-fold lower than in human islets (Fig. 4).

View larger version (10K): [in this window] [in a new window]   Figure 1. Schematic protocol for generating expanded populations of human islet-derived precursor cells (hIPCs) and their differentiation to functional islet-like cells aggregates (ICAs). The protocol involves a 4-day in vitro differentiation of hIPCs into epithelial cell clusters that mature in vivo into functional ICAs following transplantation under the kidney capsules of nonobese diabetic/severe combined immunodeficient mice.

View larger version (36K): [in this window] [in a new window]   Figure 2. Serum human and mouse C-peptide levels in euglycemic nonobese diabetic/severe combined immunodeficient mice after transplantation of human islet-derived precursor cell epithelial cell clusters under their left kidney capsules. (A): Mouse and human C-peptide levels in samples taken before (0 minutes) and 30 minutes after glucose (2 mg/g) was administered intraperitoneally to a total of 34 mice transplanted 12–24 days earlier. The bars represent mean ± SE (* p < .03; ** p < .0001). (B): Human C-peptide levels in individual samples taken before (0 minutes) and 30 minutes after glucose (2 mg/g) was administered intraperitoneally in 22 of 34 mice transplanted 12–24 days before that responded to glucose. These are the same mice as shown in (A). Abbreviation: min, minutes.

View larger version (43K): [in this window] [in a new window]   Figure 3. Immunostaining showing expression of human C-peptide and glucagon in mature human islet-derived precursor cell islet-like cell aggregates recovered from two kidney grafts after 1 and 3 months in vivo. Arrowheads indicate examples of coexpression of glucagon and C-peptide (yellow). Arrows demonstrate examples of cells stained for C-peptide (green, enlarged in the inset). The yellow dashed line delineates the border of the graft and kidney. Nuclei were stained with 4,6-diamidino-2-phenylindole (blue). Original magnification, x25. Scale bar = 50 µm.

View larger version (11K): [in this window] [in a new window]   Figure 4. Insulin (A), glucagon (B), and somatostatin (C) mRNA levels in epithelial cell clusters (ECCs) on the day of transplant (day 0) in transplants retrieved after the indicated days in vivo. Human islet-derived precursor cell ECCs were transplanted under the left kidney capsules of nonobese diabetic/severe combined immunodeficient mice and allowed to differentiate and mature in vivo. The data points for retrieved transplants represent measurements of mRNA levels from individual mice. All polymerase chain reaction results were normalized to human glyceraldehyde-3-phosphate dehydrogenase mRNA content and expressed as a percentage of the average transcript level in three preparations of human islets.

View larger version (83K): [in this window] [in a new window]   Figure 5. Human islet-derived precursor cells (hIPCs) are capable of differentiating to adipocytes, chondrocytes, and osteocytes and express CD105, CD73, and CD90. Photomicrographs showing oil red O staining of hIPCs after differentiation to adipocytes (A), von Kossa staining after differentiation to osteocytes (B), and Alcian Blue staining after differentiation to chondrocytes (C). Expression of cell surface markers CD90, CD73, and CD105 was assessed in proliferating hIPCs at passage 10. In each panel, cells treated with isotype control antibody are indicated with a dotted line, and hIPCs or bone marrow-derived MSCs treated with CD marker-specific antibodies are indicated with solid or dashed lines, respectively (D). Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

View larger version (14K): [in this window] [in a new window]   Figure 6. CD105+/CD73+/CD90+ cells sorted from adult human islets express insulin mRNA. Comparisons of the levels of insulin mRNA in groups of 10, 20, 40, 80, 160, 320, and 640 CD105/CD73/CD90-positive and -negative cells from freshly isolated islets. The levels of insulin mRNA were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNAs. The results are from five islet preparations and, for each preparation, the average normalized level of insulin mRNA in CD105/CD73/CD90-negative cells was set at 1.0. Individual values for both negative and positive cells were calculated relative to this average value.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

The origin of hIPCs has been controversial. In our initial paper [10], we presented evidence consistent with the idea that hIPCs were derived by epithelial-to-mesenchymal transition (EMT) from insulin-expressing cells. It is important to note that we did not conclude that we had proved this hypothesis and acknowledged that cell lineage analysis was needed. Perhaps the most convincing evidence for EMT was our finding that cells that expressed both insulin transcript and vimentin peptide, a marker of mesenchymal cells, appeared in cultures of adult human islets during the initial 14 days [10]. In agreement with this observation is our finding that fluorescent beta cells from MIP-GFP mice [21] migrate out from mouse islets as mesenchymal-appearing cells [11] and that of Weinberg et al. [14] in which mouse fibroblast-like cells marked as beta cells persisted in culture for several weeks but did not proliferate. Thus, mouse beta cells appear capable of transitioning to a mesenchymal phenotype in culture but do not persist. In attempts to determine whether mouse IPCs that proliferate in culture are derived from beta cells, four groups have performed lineage tracing analyses with cells derived from transgenic/knockout mouse models. Chase et al. [12], Weinberg et al. [14], Atouf et al. [13], and we [11] found no evidence that mesenchymal-appearing cells in long-term mouse IPC cultures were derived from beta cells. We do not know, however, whether derivation of mouse IPCs is from the same source as hIPCs. However, it is not obvious how to perform lineage analysis of cells from human islets. It is, of course, possible that MSCs that give rise to hIPCs are found in human islets in situ. We have begun to study this possibility by attempting to quantify and isolate CD105⁺/CD73⁺/CD90⁺ cells directly from islets that have not been cultured in vitro. In six preparations, we found that CD105⁺/CD73⁺/CD90⁺ cells make up approximately 1.2% of cells isolated from islets prior to their incubation in culture medium. Unlike bone marrow-derived MSCs, these CD105⁺/CD73⁺/CD90⁺ cells from human islets express low levels of insulin mRNA (Fig. 6). Moreover, CD105⁺/CD73⁺/CD90⁺ cells expanded after sorting from early human islet cultures and proliferative populations of hIPCs both exhibit epigenetic marks at the insulin gene that are characteristic of genes that are being actively transcribed [20]. Thus, it is possible that hIPCs are derived from MSCs that are present in islets in situ.

The idea that mesenchymal cells derived from adult tissues, like cells from fetal pancreas [22], may serve as endocrine pancreas precursor cells has been suggested by others also. Mesenchymal cells that can be transitioned/differentiated to express islet hormones have been derived from rodent [23–26] and human [27–29] bone marrow (BM-MSCs), human pancreatic ductal epithelium [30], human adipose tissue [31], and human islets [32, 33]. Human BM-MSCs were found to express insulin and other islet hormones after manipulation of the in vitro culture conditions [27] or after ectopic expression of transcription factors such as PDX-1/IPF-1 [27–29]. In some of these studies, glucose-stimulated insulin release in vitro and insulin secretion after transplantation into immunocompromised mice were observed [28, 29]. Similar transitions have been found in BM-MSCs from mice [24, 26] and rats [23, 25]. MSCs that could be made to express insulin by changing the culture conditions have been derived from human adipose tissue [31] and MSCs that could express islet transcription factors from pancreatic ductal epithelium [30]. MSCs from human islets similar to hIPCs have been reported by others. Eberhardt et al. immortalized human islet-derived MSCs by infection with lentiviral vectors encoding telomerase and mBmi and showed that these cells could express insulin, glucagon, and somatostatin [32]. Gallo et al. [33] derived MSCs from human islets that were made to transition to islet-like clusters under serum-free conditions, using a protocol very similar to ours [10], and showed that these cells express and release insulin.

We suggest there are at least four stages in vitro and in vivo in the development of mesenchymal hIPCs to functional ICAs in mice during our protocol. These include (a) exponential proliferation of mesenchymal precursor cells, (b) initial differentiation of mesenchymal cells into epithelial cells, (c) differentiation of epithelial cells into hormone-producing pancreatic endocrine cells, and (d) maturation of endocrine cells into cells that contain high levels of pancreatic hormones and exhibit glucose-stimulated insulin secretion. We showed previously that hIPCs could undergo the first three of these stages in vitro [10]. However, our previous efforts to induce functional maturation of these cells in vitro required culturing of hIPCs for up to 21 days in differentiation medium devoid of growth/survival factors. This led to death of the majority of the cells, and the few that remained expressed insulin mRNA at levels less than 0.02% of that in human islets. We have, therefore, pursued a methodology to improve the efficiency of differentiation and maturation of endocrine cells and achieve levels of human insulin in the range needed for maintenance of normal glucose homeostasis. As described herein, our new method includes in vitro proliferation of mesenchymal hIPCs in growth medium containing 10% fetal bovine serum for approximately 50 days to allow a 1,000-fold or greater expansion followed by in vitro differentiation of mesenchymal cells into cell clusters that exhibit an epithelial phenotype (ECCs) after only 4 days in a defined differentiation medium without serum. (In a preliminary experiment, we have shown that hIPCs expanded at least 100,000-fold will differentiate and mature also.) These ECCs are then transplanted into mice where they undergo further differentiation into endocrine cells expressing insulin, glucagon, and somatostatin. The functional maturation of some of these cells is characterized by the expression of glucokinase, glucose transporter-2, and glucagon-like peptide-1 receptor and the demonstrated capacity for glucose-responsive insulin secretion. It is important to note that the levels of hormone expression in these cells are still below those found in normal islets, and we have not demonstrated that these transplanted cells are capable of maintaining normal glucose homeostasis. Nevertheless, we have found that two important stages in the development of hIPCs into more mature endocrine pancreas cells can be achieved in vivo and may not require novel in vitro conditions.

The results of the studies described here show that hIPCs are a type of MSC that may represent an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet-like cells for replacement therapy in type 1 diabetes. With regard to possible replacement therapy in humans, it is important to note that, even when mice that had received hIPC grafts were maintained for up to 6 months, we found no evidence for local or distal tumor formation. In the accompanying paper, we have found that the insulin gene in both early and late passage hIPCs exhibits chromatin modifications on histones H3 and H4 that are characteristic of active genes and that are also found at the same locus in fresh human islets [20]. Importantly, neither human bone marrow MSCs nor HeLa cells exhibit these chromatin modifications at the insulin gene. Thus, although hIPCs appear to be a type of MSC, the epigenetic marking of the insulin gene in hIPCs may reflect a unique endocrine commitment for these precursor cells that distinguishes them from other MSCs derived from adult tissues. Lastly, although we have not shown that the CD105⁺/CD73⁺/CD90⁺ cells from freshly isolated human islets are the source of expanded populations of hIPCs, it is possible that hIPCs are derived from MSC precursor cells that are present in adult islets in situ.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported by the Intramural Research Program, NIDDK, NIH. We thank both the National Islet Cell Resource Program (Duarte, CA) and our colleagues at NIDDK, Drs. David Harlan and Eric Liu, for supplying human islets. B.D. and L.I. contributed equally to this work.

	REFERENCES

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