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First published online July 5, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2837 -2844
doi:10.1634/stemcells.2007-0164; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Generation of Insulin-Producing Cells from Human Bone Marrow Mesenchymal Stem Cells by Genetic Manipulation

Ohad Karnielia, Yael Izhar-Pratoa, Shlomo Bulvikb, Shimon Efrata

aDepartment of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel;
bLaniado Medical Center, Nethanya, Israel

Key Words. β Cell replacement • Insulin secretion • Pancreatic duodenal homeobox 1 • Cell transplantation

Correspondence: Shimon Efrat, Ph.D., Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel. Telephone: 972-3-640-7701; Fax: 972-3-640-9950; e-mail: sefrat{at}post.tau.ac.il

Received on March 7, 2007; accepted for publication on June 26, 2007.

First published online in STEM CELLS EXPRESS  July 5, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
β Cell replacement is a promising approach for treatment of type 1 diabetes; however, it is limited by a shortage of pancreas donors. The pluripotent MSC in adult bone marrow (BM) offer an attractive source of stem cells for generation of surrogate β cells. BM-MSC can be obtained with relative ease from each patient, allowing potential circumvention of allograft rejection. Here, we report a procedure for expansion of BM-MSC in vitro and their differentiation into insulin-producing cells. The pancreatic duodenal homeobox 1 (Pdx1) gene was expressed in BM-MSC from 14 human donors, and the extent of differentiation of these cells toward the β-cell phenotype was evaluated. RNA and protein analyses documented the activation of expression of all four islet hormones. However, the cells lacked expression of NEUROD1, a key transcription factor in differentiated β cells. A significant insulin content, as well as glucose-stimulated insulin release, were demonstrated in vitro. Cell transplantation into streptozotocin-diabetic immunodeficient mice resulted in further differentiation, including induction of NEUROD1, and reduction of hyperglycemia. These findings were reproducible in BM-MSC from 9 of 14 donors of both sexes, ages 19–62. These results suggest a therapeutic potential for PDX1-expressing BM-MSC in β-cell replacement in patients with 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
 
β Cell replacement represents the most promising approach for treatment of type 1 diabetes; however, it is severely limited by a shortage of pancreas donors. The pluripotent MSC in adult bone marrow (BM), which have been shown to differentiate in vivo into a variety of ectodermal, mesodermal, and endodermal tissues in both mice [1, 2] and humans [3, 4], offer an attractive source of stem cells for generation of surrogate β cells. BM-MSC can be obtained with relative ease from each patient, allowing potential circumvention of allograft rejection. Previous work has suggested that grafted mouse BM cells could spontaneously differentiate in vivo into endocrine pancreas cells [5]; however, additional studies were unable to support these findings [6–8]. Rather, it was proposed that both mouse [9] and human [10] BM-MSC reversed hyperglycemia in streptozotocin (STZ)-treated mice by inducing regeneration of endogenous islets. Manipulation of culture conditions was reported to induce low levels of insulin in rodent BM-MSC [11–14]; however, the stability of this phenotype was unclear.

We have recently shown that stable expression of pancreatic duodenal homeobox 1 (PDX1), a transcription factor that plays key roles in pancreas development and β-cell gene expression, in cultured human fetal liver cells activates a phenotype close to that of differentiated β cells [15, 16]. These cells produced and stored mature insulin in amounts up to 60% of those produced by normal β cells, released it in response to physiological glucose levels, and replaced β-cell function in STZ-diabetic immunodeficient mice. These findings demonstrate the feasibility of inducing a profound phenotypic change in cultured human tissue progenitor cells by expression of a single master regulator gene. Other reports demonstrated the inductive effects of ectopic PDX1 expression [17–22]; however, most of them involved cells from endodermal origin. Introduction of transcription factor genes into cultured human BM cells was reported to activate a number of β-cell genes [23, 24]; however, the reproducibility of these results in multiple donors was not reported, and no functional studies in vivo were performed. Here, we evaluated the extent of the phenotypic change induced by stable expression of PDX1 in cultured BM-MSC from 14 human donors, both males and females, ages 19–70. RNA and protein analyses documented the activation of expression of all four islet hormones. However, the cells lacked expression of NEUROD1, a key transcription factor in differentiated β cells. A significant insulin content, as well as glucose-stimulated insulin release, was demonstrated in vitro. Cell transplantation into STZ-diabetic immunodeficient mice resulted in further differentiation, including induction of NEUROD1, and reduction of hyperglycemia. These findings were reproducible in BM-MSC from 9 of 14 donors. These results suggest a therapeutic potential for PDX1-expressing BM-MSC in cell therapy of diabetes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
Human BM cells were obtained from adult donors at the Laniado Hospital under approved protocols. They were cultured essentially as described [25]. Briefly, 10-ml BM aspirates were taken from the iliac crest of male and female donors between the ages of 19 and 70. Mononuclear cells were isolated using a density gradient (Ficoll-Paque; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and resuspended in {alpha}-minimal essential medium containing 25 mM glucose (all culture medium components were from Biological Industries [Beth Haemek, Israel, http://www.bioind.com] unless otherwise indicated) and supplemented with 16% fetal bovine serum (lot no. CPB0183; HyClone, Logan, UT, http://www.hyclone.com), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. Cells were plated in 10-cm culture dishes (Corning Enterprises, Corning, NY, http://www.corning.com) and incubated at 37°C with 5% humidified CO2. In addition, the medium was also supplemented with 10 ng/ml human basic fibroblast growth factor (bFGF) (Cytolabs, Rehovot, Israel, http://www.cytolabs.com) to promote cell proliferation. After 24 hours, nonadherent cells were discarded, and adherent cells were thoroughly washed twice with phosphate-buffered saline (PBS). The cells were incubated for 5–7 days, harvested by treatment with 0.25% trypsin and 1 mM EDTA for 5 minutes at 37°C, seeded at 50–100 cells per cm2, and cultured to confluence. They were then continuously maintained by subculturing at a similar density.

Fluorescence-Activated Cell Sorting Analysis
Cells seeded at 50–100 cells per cm2 and grown to confluence were harvested with trypsin, washed twice with PBS, and incubated with labeled antibodies for 30 minutes in PBS at 4°C in the dark. They were washed twice, resuspended in PBS, and analyzed using a FACSort instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). All antibodies were mouse anti-human monoclonals from DakoCytomation (Glostrup, Denmark, http://www.dakocytomation.com): anti-CD-34-fluorescein isothiocyanate (FITC), anti-CD-45-FITC, anti-CD-105-phycoerythrin (PE), anti-CD-29-FITC, anti-CD-90-FITC, anti-CD14-PE, and anti-CD-44-PE, at the dilutions indicated by the manufacturer.

Differentiation into Bone and Adipose Cells
BM-MSC propagated for 3 weeks in culture were incubated in osteogenic or adipogenic differentiation medium [26] for 21 days and stained with alizarin red or oil red, respectively, as described [26]. Nuclear DNA was stained with 4,6-diamidino-2-phenylindole (DAPI).

Retrovirus Production
The rat Pdx1 cDNA was cloned between the BamHI and SalI restriction sites of the pBabe-hygromycin vector (Addgen, Cambridge, MA, http://www.addgene.org). Virus was produced in 293T cells by cotransfection with the PCL-Ampho plasmid (Imgenex, San Diego, CA, http://www.imgenex.com). Twenty-four hours post-transfection, the medium was replaced with fresh medium. The medium containing the virus was harvested 2–4 days later.

Cell Infection and Differentiation
Cells (3 x 105) were plated in a 10-cm culture dish in medium supplemented with 20% conditioned medium from a highly proliferative BM-MSC culture [27] for 24 hours. Cells were infected with freshly harvested virus at a multiplicity of infection of 10:1 overnight. The infection was repeated two more times in the following days. Selection was initiated 2–3 days later with 50 µg/ml hygromycin for 5 days in the absence of conditioned medium. Following recovery for 6 days, the selection was repeated. Following selection (a total of 21 days from infection), the cells were switched to differentiation medium lacking bFGF, grown to confluence, and split 1:3 to approximately 5 x 105 cells per 10-cm dish. They were thereafter split 1:3 every 7–10 days.

Quantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated using the Versagene RNA Isolation Kit (Gentra, Minneapolis, MN, http://www.qiagen.com) and treated with DNase according to the manufacturer's protocol. cDNA was prepared using Superscript III (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). cDNA samples derived from 50 ng of total RNA were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using the TaqMan primers and probes listed in Table 1(Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) in an ABI 7300 Real-Time PCR System (Applied Biosystems). The results were normalized to human large ribosomal protein P0 cDNA. For the analysis in Figure 3, the following TaqMan primers and probes were used: Hs00357871_s1 for INSAM1, Hs00606262_g1 for HDAC1, Hs00187320_m1 for HDAC3, and Hs00277039_m1 for CCND1.


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  		Table 1. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of islet gene expression in BMP cells

 
Microarray Analysis
BM-MSC from three male donors (ages 19, 23, and 26) were infected with PDX1– or empty virus in duplicate plates. Total RNA was isolated following 34 days of differentiation. The 12 RNA samples were amplified and hybridized to the Affymetrix human U133 array (Santa Clara, CA, http://www.affymetrix.com), using the manufacturer's protocol. The array data were analyzed using dChip software and normalized using invariant set normalization. Present gene thresholds were set at four of the six replicate chips in each treatment. Changes greater than twofold were determined with p < .05.

Immunofluorescence
Cells were seeded on glass slides in a 24-well dish and fixed in 4% paraformaldehyde in PBS. Blocking and antibody binding were performed in 10% fetal calf serum, 1% bovine serum albumin, and 0.2% saponin in PBS. Antibody dilutions were as follows: mouse anti-insulin (1:200) (Sigma-Aldrich), goat anti-glucagon (1:100) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com; does not recognize glucagon-like peptide 1), rabbit anti-somatostatin (1:500) (DakoCytomation), mouse anti-heat shock protein (anti-HSP) 27 (1:50) (Lab Vision, Fremont, CA, http://www.labvision.com), rabbit anti-PDX1 (1:5,000) (a gift from C. Wright), rabbit anti-pancreatic polypeptide (PP) (1:200) (Linco, St. Charles, MI, http://www.lincoresearch.com), and mouse anti-human C-peptide (1:50) (BioDesign, Saco, ME, http://meridianlifescience.com). Secondary antibodies were Cy3 anti-mouse (1:200), Cy3 anti-rabbit (1:500), Cy2 anti-goat (1:100), and Cy2 anti-mouse (1:100) (all from Biomeda, Foster City, CA, http://biomeda.com). Nuclear DNA was stained with DAPI. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were rehydrated, blocked, and stained as described [16].

Insulin and C-Peptide Assays
For insulin secretion assays cells were preincubated for 1 hour in Krebs-Ringer buffer (KRB), followed by incubation for 30 minutes in KRB containing 0.5 mM 1-isobutyl-3-methylxanthine and glucose at the indicated concentrations. The buffer was collected, and the cells were extracted in 3 M acetic acid. The amount of insulin in the buffer and cell extract was determined by ultrasensitive insulin enzyme-linked immunosorbent assay (ELISA) (Mercodia, Uppsala, Sweden, http://www.mercodia.com) according to the manufacturer's protocol. Serum insulin was measured with the same assay. Serum human C-peptide was assayed using the human-specific C-peptide ELISA (Marcodia, Sweden) following the manufacturer's protocol. Serum mouse C-peptide was assayed using mouse-specific C-peptide-1 ELISA (Alpco Diagnostics, Salem, NH, http://www.alpco.com).

Cell Transplantation
Six- to 10-week-old severe combined immunodeficient (SCID) female mice (Harlan, Jerusalem, Israel, http://www.harlan.com) were made hyperglycemic by a single i.p. injection of 200 µg of STZ per g of body weight. When blood glucose reached levels over 300 mg/dl, within 3–7 days of STZ injection, mice were anesthetized with an i.p. injection of 15 µl of 2.5% avertin per g of body weight, and 3 x 106 cells were transplanted under the left kidney capsule. Blood glucose levels were monitored twice a week in samples obtained from the tail vain using Accutrend strips (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Serum C-peptide levels were determined in samples obtained from the orbital plexus. For the glucose tolerance test, mice fasted for 6–10 hours were injected i.p. with 1 mg of glucose in saline per g of body weight. Blood glucose levels were monitored at the indicated time points in samples obtained from the tail vein. At the end of the experiment, the mice were sacrificed, and the kidney was removed for histological or RNA analyses. In some mice, survival nephrectomy was performed, and the mice were monitored 1 day later for changes in blood glucose levels. The animal protocols were approved by the Tel Aviv University Institutional Animal Care and Use Committee.

Statistical Analysis
Significance was determined using Student's t test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
BM-MSC were obtained from BM samples collected from adult human donors by plating mononuclear cells in tissue culture dishes and propagation of adherent cells. Following 28 days in culture, the vast majority of cells stained for the markers CD-29 (97.5%), CD-44 (92.3%), CD-90 (98.3%), and CD-105 (96.9%), and only a small minority manifested expression of the markers CD-14 (0.03%), CD-34 (1.5%), and CD-45 (0.77%). Taken together, this marker combination is widely accepted as a signature of BM-MSC [23, 28–30]. According to these markers, the cell population at this time point was quite uniform. In addition, expression of CD106 and CXCR4, shown to be expressed in BM-MSC [28, 31], and the absence of C-KIT and CD11B [25] were documented in our cultures by reverse transcription (RT)-PCR and immunostaining (data not shown). To demonstrate their differentiation pluripotency, the BM-MSC were subjected to conditions known to induce differentiation into bone or adipose cells. The results showed a uniform adipogenic differentiation in >90% of the cells and a massive osteogenic differentiation. Typically, a 10-ml BM sample yielded approximately 3 x 103 BM-MSC. These cells were expanded within 30 days to 4 x 108 cells, representing approximately 17 population doublings at an average doubling time of 42 hours.

To activate the islet cell phenotype in these cells, cells expanded for 20 days in culture were infected with a retrovirus expressing rat Pdx1 cDNA under control of the viral long terminal repeat. The vector included a hygromycin-resistance gene under control of the phosphoglycerate kinase promoter. The efficiency of infection of BM-MSC with this vector ranged between 40% and 70%. Following selection, the surviving cells (bone marrow cells harboring PDX1 virus [BMP]) were expanded for approximately six population doublings before further analysis. Control cells were infected with an empty vector lacking the Pdx1 insert but containing the hygromycin-resistance gene, and the surviving cells (bone marrow cells harboring empty virus [BME]) were expanded and studied in parallel to BMP cells. Approximately 3 weeks after viral infection, the doubling time of BMP cells increased to 3–4 days, whereas BME cells continued to divide approximately once in 2 days. Similar expansion rates before and after PDX1 expression were observed among cells from three different male donors ages 19–39, which were studied in detail for growth properties.

qRT-PCR analysis of RNA from BME cells obtained from three different donors detected low levels of expression of a number of islet genes, including somatostatin, PP, PAX6, NKX2.2, NKX6.1, and proconvertase (PC) 1/3 (Table 1). Expression of ISL1 was more pronounced.

Expression of PDX1, demonstrated by immunofluorescence in all BMP cells (Fig. 1), activated the expression of multiple islet genes. Insulin expression was detected in 40%–60% of the cells expressing PDX1, depending on the donor (Fig. 1). Overall, insulin transcripts were detected in BMP cells from 9 of the 14 donors studied, including 10 male and 4 female donors, ages 19–70 (Table 2). Insulin mRNA was first detected in BMP cells from two donors starting from 7 days after Pdx1 gene transfer, whereas in the other seven donors this transcript first appeared 28 days postinfection. Significant amounts of insulin mRNA accumulated in cells from all nine donors by 40 days from infection. In contrast, all BMP cells were positive for glucagon (Fig. 1). Significant levels of glucagon mRNA were detected starting from 14 days postinfection in BMP cells from 11 of the 14 donors studied (Table 2). Somatostatin staining was also detected in all the cells (Fig. 1), and its transcripts were present in significant levels starting from 14 days postinfection in 12 of 14 donors (Table 2). A weak PP staining was also detected in all BMP cells, as well as in BME cells (data not shown), and its transcripts were present in significant levels in both BME cells and in BMP cells from 12 of 14 donors (Table 2). BME cells did not stain for insulin, glucagon, or somatostatin (data not shown).


Figure 1
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  		Figure 1. Expression of islet hormones in BMP cells. BMP cells were differentiated for 24–34 days and stained with the indicated antibodies. Nuclei were stained blue with 4,6-diamidino-2-phenylindole. No staining for the four antigens was detected in BME cells. Scale bar = 20 µm.

 


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  		Table 2. Variability in expression of islet hormone transcripts among BMP from different donors

 
In addition to islet hormones, expression of PDX1 activated the expression of glucokinase, the transcription factor HNF6, and low levels of GLUT2, PAX4, and HLXB9 transcripts, as judged by qRT-PCR analysis (Table 1). Expression of PAX6, NKX2.2, NKX6.1, and ISL1 transcripts was induced severalfold, compared with the basal levels seen in BME cells (Table 1). In contrast, transcripts for NEUROD1, NGN3, SUR1, KIR6.2, and PC2 were not detected in BMP cells from most donors (Table 1). Furthermore, the rat PDX1 used here did not activate transcription of the endogenous human PDX1 gene (Table 1).

Microarray analyses performed with RNA extracted from cells derived from three donors 24–34 days following initiation of differentiation detected a >2x increase in expression of 126 genes, and a total of 412 genes were downregulated >2x in BMP cells, compared with BME cells (the complete list is given in the supplemental online data). This analysis detected expression in BME cells of a number of genes encoding components of secretory vesicles, including syntaxin 3, synaptobrevin 1, and synaptotagmin 1, in addition to the islet genes detected by qRT-PCR (Table 1). The inability to detect expression of PP transcripts in BME cells may be due to a low affinity of the PP probes on this microarray. The microarray analysis detected the activation of a gene expressed in pancreatic exocrine cells, amylase, in BMP cells. No change in expression of the BM-MSC markers listed above was observed between BME and BMP cells by the microarray analyses.

The absence of expression of NEUROD1 in BMP cells is likely a major cause for the incomplete β-cell phenotype observed in these cells. The microarray data, and their confirmation by qRT-PCR analysis (Fig. 2), revealed a pronounced upregulation of transcripts encoding repressors of NEUROD1 gene transcription, insulinoma-associated 1, histone deacetylases 1 and 3, and cyclin D1 [32], in BMP cells compared with BME cells.


Figure 2
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  		Figure 2. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of expression of repressors of NeuroD1 gene transcription. RNA was extracted from BMP cells from three different donors 24–34 days following shift to differentiation medium and analyzed by qRT-PCR for the indicated genes. Values are fold change of BMP/BME ± SD (n = 3). Abbreviations: CCND1, cyclin D1; HDAC1, histone deacetylase 1; HDAC3, histone deacetylase 3; INSM1, insulinoma-associated 1.

 
To evaluate the functional differentiation of BMP cells, they were tested for insulin content, glucose-induced insulin secretion in vitro, and the ability to replace β-cell function in vivo. Insulin content of BMP cells from different donors ranged between 27 and 90 ng per 106 cells. A comparison to normal islets is somewhat difficult, as different papers cite values ranging between 1 and 15 mg per human pancreas, which contains approximately 109 islet cells. Assuming an average value of 10 mg per 109 cells, the maximal insulin content observed in BMP cells in vitro represents approximately 1% of the content of normal human islets [33, 34]. Insulin secretion from BMP cells was regulated by glucose; however, it occurred in response to glucose concentrations above the physiological range (Fig. 3). Although most of the response of normal human islets occurs between 5 and 10 mM glucose, BMP cells secreted most of the insulin in response to 12–20 mM glucose. Cells from all three donors tested showed a response to glucose, ranging between 5.3-fold and 13.7-fold.


Figure 3
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  		Figure 3. Glucose-induced insulin secretion in BMP cells. BMP cells derived from three donors were tested 24–34 days following shift to differentiation medium. Cells were incubated for 30 minutes in Krebs-Ringer buffer containing 0.5 mM 1-isobutyl-3-methylxanthine and glucose at the indicated concentration in triplicate. The amount of insulin in the buffer and cell extract was determined by enzyme-linked immunosorbent assay. Values are mean ± SD.

 
Transplantation of BMP cells under the renal capsule of STZ-diabetic SCID mice resulted in reduction of blood glucose levels, beginning 12 days post-transplantation, from over 300 mg/dl to approximately 200 mg/dl at 40 days post-transplantation (Fig. 4A). Blood glucose levels were then stabilized for the remainder of the follow-up period, 5 weeks later. In contrast, in untransplanted mice, mice transplanted with unmanipulated BM-MSC (data not shown), and mice transplanted with BME cells (Fig. 4A), blood glucose levels continued to rise to over 600 mg/dl, and most mice died within the first month following STZ treatment. Glucose tolerance test performed 6–8 weeks following transplantation revealed that BMP cells were able to respond to the glucose challenge, although their response was not as efficient as that of normal islets (Fig. 4B). In contrast, BME cells did not show any response to glucose challenge in vivo (Fig. 4B). Serum human C-peptide levels in mice transplanted with BMP cells measured 55 days post-transplantation ranged between 0.06 and 0.39 ng/ml, whereas no human C-peptide was detected in mice transplanted with BME. Mouse C-peptide in all but one of the mice studied was below the assay detection limit. In a single mouse, its levels were 0.72 ng/ml, which could represent residual β cells not destroyed by STZ (normal mice showed values around 1.7 ng/ml). One mouse transplanted with BMP cells and kept for a longer follow-up period showed fed blood glucose levels of 131 mg/dl 140 days post-transplantation. The serum human C-peptide level at this time point was 1 ng/ml. Nephrectomy in this mouse resulted in restoration of hyperglycemia (357 mg/dl) the following day. These findings indicate that human BM cells did not induce significant regeneration of mouse islets in the transplanted mice, even after a long period of time. This conclusion is supported by the detection of very few insulin-positive cells in sections of the pancreas removed from the transplanted mice at the end of the experiment and the absence of cells positive for the human marker HSP27 in those sections, indicating that the transplanted BMP cells did not migrate from the kidney to the pancreas (data not shown). Similar low numbers of insulin-positive cells were found in the pancreas of mice transplanted with BME cells, which remained hyperglycemic. Histological analysis of the kidneys removed at the end of the experiment revealed an intense staining for human C-peptide and insulin in the vast majority of transplanted cells, which were positively identified as human cells by staining for human HSP27 (data not shown), and a very weak glucagon staining in a small number of cells (Fig. 5). RNA was extracted from a kidney transplanted with BMP cells, which was removed 140 days post-transplantation and analyzed by qRT-PCR for human-specific transcripts. The analysis revealed a 19-fold increase in insulin mRNA levels, compared with the cultured BMP cells (Fig. 5E). Glucagon, somatostatin, and PP transcripts were downregulated five-, six-, and twofold, respectively (Fig. 5F). Notably, human PDX1 and NEUROD1 transcripts, as well as transcripts encoding the KATP+ channel subunits SUR1 and KIR6.2, were induced in vivo from undetectable levels in vitro (Fig. 5G). In addition, transcripts encoding GLUT2 and glucokinase were upregulated eight- and threefold, respectively. Taken together, these findings demonstrate significant BMP cell differentiation in vivo, manifested by induction of key transcription factors and regulators of glucose sensing, an increase in insulin expression, and a decrease in non-β-cell islet hormones.


Figure 4
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  		Figure 4. Function of BMP cells in vivo. BMP and BME cells 24–34 days following shift to differentiation medium were transplanted into streptozotocin-diabetic severe combined immunodeficient mice. (A): Fed blood glucose levels (mean ± SD). BMP and BME values were significantly different from day 21 on (p < .05). (B): Glucose tolerance test performed on mice in (A) 6–8 weeks following transplantation, compared with a normal (nondiabetic) control. Values are mean ± SD.

 


Figure 5
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  		Figure 5. Analysis of BMP cells transplanted under the renal capsule. (A–D): Immunofluorescence of kidneys removed 6 weeks post-transplantation from two mice in the experiment shown in Figure 4. Tissue sections were stained with the indicated antibodies. Nuclei were stained blue with 4,6-diamidino-2-phenylindole. Scale bar = 20 µm (A, B, D) and 40 µm (C). (A) and (B) are from the same field; (D) is a magnification of the marked area in (C). (E–G): Quantitative reverse transcription-polymerase chain reaction analysis of RNA extracted from kidney removed 140 days post-transplantation, using human-specific primers. (E): Genes upregulated in vivo; (F): Genes downregulated in vivo; (G): Genes not expressed in vitro and induced in vivo. {Delta}Ct represents the difference between the cycle threshold of the assayed gene and that of the normalizing gene. Abbreviation: PP, pancreatic polypeptide.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
We report a procedure for expansion of BM-MSC in vitro and their differentiation into insulin-producing cells. Our findings demonstrate that PDX1 induces a profound change in gene expression in an adult human mesodermal tissue cultured in vitro and can drive its differentiation a long way toward the β-cell phenotype. These findings were reproducible in cells from nine donors of both sexes, ages 19–62. Overall, 538 genes were up- or downregulated by PDX1 >twofold in BM-MSC. Since the number of known direct targets of PDX1 is much smaller, this finding suggests that expression of many of these genes was affected indirectly. These changes were sufficient for activation of expression of all islet hormones, as well as an exocrine marker, in BM-MSC. However, insulin expression was detected in only approximately half of the cells, whereas the other three islet hormones were present in all cells. This finding may reflect heterogeneity within the BM-MSC population, although judging from the cell surface markers analyzed the population was quite uniform. The relatively long lag for appearance of insulin transcripts in the cells following PDX1 expression indicates that additional events are involved in the mechanism of activation of this gene in BM-MSC. Expression of one of the key transcription factors required for insulin gene expression and β-cell function, NEUROD1, could not be detected in the differentiated cells in vitro. This result could be explained by the finding that PDX1 expression upregulated transcripts for insulinoma-associated 1, histone deacetylases 1 and 3, and cyclin D1, which were shown to act as repressors of NEUROD1 expression [32]. The pattern of transcription factors detected in BMP cells (Table 1), including PAX4, PAX6, NKX2.2, NKX6.1, HLXB9, HNF6, and ISL1 but lacking NGN3 and NEUROD1, does not entirely match the accepted hierarchy of transcription factors expressed in normal islet development. However, it should be noted that the data presented reflect the situation at the time of analysis, when insulin was detected. It is possible that certain transcription factors were briefly induced at earlier time points but were subsequently downregulated. BMP cells represent a useful model in which the kinetics of expression of the transcription factors involved in this in vitro differentiation process can be studied.

Another prominent deviation of BMP cells from the normal β-cell phenotype is the lack of expression of the genes encoding the KATP+ channel subunits, SUR1 and KIR6.2. Nevertheless, BMP cells manifest a glucose-stimulated insulin secretion of more than 13-fold, although the response is markedly shifted to the right. Induction of GLUT2 and GK in BMP cells (Table 1) likely contributes to their glucose responsiveness, but the absence of a KATP+ channel may be responsible for the shift to the right. The observed response is unlikely to reflect an abnormally high ratio of HK/GK or GLUT1/GLUT2 expression, which would be expected to cause a shift to the left. A fourfold induction in PC1/3 transcripts (Table 1) suggests the occurrence of normal proinsulin processing; however, in the absence of PC2, this possibility should be confirmed by high-performance liquid chromatography analyses.

Cell transplantation in vivo resulted in further differentiation, as demonstrated by immunofluorescence and qRT-PCR analyses. A prominent change occurred in the pattern of hormone expression. Coexpression of islet hormones in BMP cells in vitro is reminiscent of the hormone coexpression documented in human fetal islets [35] and is a manifestation of cell immaturity. In contrast, the occurrence of insulin staining in virtually all BMP cells in vivo and the reduction in glucagon staining represent significant differentiation. In addition, RNA analyses of transplanted BMP cells detected the induction of four key genes that were not expressed in cultured BMP cells (human PDX1, NEUROD1, KIR6.2, and SUR1), as well as an increase in insulin, GLUT2, and glucokinase transcripts and a decrease in expression of the other three islet hormones. These changes are likely responsible for the ability of BMP cells to reduce glycemia in vivo and respond to a glucose challenge.

The presence of human C-peptide and the absence of detectable mouse C-peptide in the serum of the majority of transplanted mice indicate that the reduction in glycemia was due to the transplanted human cells. In addition, the absence of detectable human cells in the pancreas, the restoration of hyperglycemia upon removal of the transplanted cells by nephrectomy, and the fact that control mice transplanted with unmanipulated human BM-MSC or with BME cells containing a vector lacking Pdx1 died with severe hyperglycemia indicate that human BM-MSC did not induce significant regeneration of endogenous mouse islets when transplanted under the renal capsule. In contrast, a recent report suggested such a capacity for human BM-MSC introduced into mice by intracardiac infusion [10].

In conclusion, our findings demonstrate the therapeutic potential of adult human BM-MSC genetically manipulated by expression of Pdx1. The considerable expansion capacity of adult human BM-MSC in vitro during at least 23 population doublings allows generation of approximately 1.6 x 1010 cells from a single 10-ml sample of BM, making autologous cell therapy quantitatively feasible without further manipulation of cell growth by telomerase, which was required for expansion in vitro of other tissue stem/progenitor cells, such as those from liver [15]. Alternatively, transplantation of insulin-producing cells derived from allogeneic BM may be facilitated by cotransplantation of unmanipulated BM cells from the same donor for creating BM chimerism, thus circumventing the risks of both graft rejection and recurring autoimmunity [36]. Expression of some islet genes in unmanipulated BM-MSC indicates a predisposition for differentiation toward islet-cell phenotype, given appropriate stimuli. Treatment of BMP cells with agents that stimulate β-cell differentiation [16] may further advance their phenotype toward that of normal β cells.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Christopher Wright for PDX1 antibodies, Amiel Dror for assistance with tissue sectioning, and Michal Zalzman for helpful discussions. This work was supported by grants from the Juvenile Diabetes Research Foundation, the Russell Berrie Foundation, and D-Cure, Diabetes Care in Israel (to S.E.).


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

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