Chromatin-Remodeling Factors Allow Differentiation of Bone Marrow Cells into Ins - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Thatava Tayarammaa, Bin Maa, Manfred Rohdeb, Hubert Mayera作者单位：aDepartment of Gene Regulation and Differentiation andbDepartment of Microbial Pathogenesis, German Research Center for Biotechnology, Braunschweig, Germany
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      【摘要】
   Type 1 diabetes is caused by the destruction of pancreatic &szlig;-cells by T cells of the immune system. Islet transplantation is a promising therapy for diabetes mellitus. Bone marrow stem cells (BMSC) have the capacity to differentiate into various cell lineages including endocrine cells of the pancreas. To investigate the conditions that allow BMSC to differentiate into insulin-producing cells, a novel in vitro method was developed by using the histone deacetylase inhibitor, trichostatin A (TSA). BMSC, cultured in presence of TSA, differentiated into islet-like clusters under appropriate culture conditions. These islet-like clusters were similar to the cells of the islets of the pancreas. The islet-like clusters showed endocrine gene expression typical for pancreatic &szlig;-cell development and function, such as insulin (I and II), glucagon, somatostatin, GLUT-2, pancreatic duodenal homeobox-1 (PDX-1), and Pax 4. Immunocytochemistry confirmed islet-like clusters contained pancreatic hormones. The colocalization of insulin and C-peptide was also observed. Enzyme-linked immunosorbent assay analysis demonstrated that insulin secretion was regulated by glucose. Western blot analysis demonstrated the presence of stored insulin. Electron microscopy of the islet-like cells revealed an ultrastructure similar to that of pancreatic &szlig;-cells, which contain insulin granules within secretory vesicles. These findings suggest that histone-deacetylating agents could allow the differentiation of BMSC into insulin-producing &szlig;-cells.
      【关键词】 Bone marrow Histone deacetylation inhibitors Trichostatin A Insulin-producing cells Diabetes5 M6 Q6 J% _  y1 {! U8 _1 b
   INTRODUCTION

Diabetes mellitus (DM) is a common metabolic disorder affecting millions of people worldwide and is characterized by abnormally high levels of glucose in blood. Type 1 diabetes (insulin-dependent) is an autoimmune disease, resulting from the body's own immune system destroying pancreatic ß-cells and causing absolute insulin deficiency. An effective treatment for Type 1 diabetes is islet cell transplantation .
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The key goals of stem cell research are to understand the manner in which differentiation is controlled and the direction that cellular differentiation will take. This should result in a better understanding of stem cell biology and allow optimal use of these cells in cell therapy. Stem cell plasticity depends on many factors, such as chromatin structure. The actual chromatin structure and its remodeling is an important mode of the epigenetic control of gene expression. The chromatin modulation includes histone acetylation and DNA methylation. Histone acetylation or DNA methylation modifiers modulate a wide variety of cellular functions, including cell differentiation. Histone deacetylase (HDAC) inhibitors are known as agents that modulate the expression of genes by increasing histone acetylation, thereby regulating chromatin structure and transcription .

We have evaluated the effect of TSA on the differentiation of BMSC into insulin-producing cells. We have found that treatment with TSA and subsequent cell culture conditions can induce the differentiation of BMSC into insulin-producing cells and other cell types typical of endocrine pancreas. Here we show that differentiated bone marrow cells express transcription factors specific for development of endocrine pancreas, such as PDX-1, hepatocyte nuclear factor (HNF)-3ß, and Pax 4. The bone marrow-derived islet-like cells secrete insulin in response to glucose stimulus. This is a novel and efficient method for the differentiation of bone marrow cells into insulin-producing cells within a short time period of 10 days. These cells could be a potentially unlimited source for the transplantation of insulin-producing cells into Type 1 diabetes patients." `5 s$ M  ^( Y# W
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MATERIALS AND METHODS. Q+ ]8 |: A% X1 g$ {' V7 y
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Mice
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C57BL/6 female mice of 6¨C10-weeks old were purchased from the Harlan Winklemann (GmbH, Borchen, Deutschland, http://www.harlan-winkelmann.de) and maintained in a specific pathogen-free facility until used. Mice were treated in accordance with institute guidelines.

BMSC Isolation and In VitroDifferentiation Conditions) v- k% g1 f! q6 A
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Bone marrow (BM) was flushed out with Dulbecco's modified Eagle's medium (DMEM) (MP Biomedicals, Germany, http://www.mpbio.com) in the presence of 10% fetal bovine serum from the tibias and femurs of C57BL/6 mice. The BM cells were plated at a density of 1 x 106 cells/cm2 in 6-well plate in normal DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml glutamine and allowed to adhere to the cell culture plate. After 12 hours of incubation, nonadherent cells were washed out with fresh serum-free DMEM medium. The cells were treated with TSA (kindly donated by Prof. Dr. J¨¹rgen Bode, GBF, Braunschweig) at a concentration of 55 nM in serum-free DMEM for 3 days. For the induction of differentiation, TSA was withdrawn from the medium, and the cells were then cultured for an additional 7 days in specific induction medium containing a 1:1 ratio of DMEM:DMEM/F12 (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 10% fetal bovine serum and 10 nM glucagon-like peptide-1 (GLP-1), in presence of high (25 mM) glucose.

Islet Isolation from Pancreas

Mouse pancreatic islets were isolated as described previously . Briefly, 6¨C10-week-old female C57BL/6 mice were killed by cervical dislocation. The pancreata were removed, without ductal injection or distention, cut into small pieces, and washed three times with ice-cold Hanks' balanced salt solution (HBSS) to remove released trypsin. The resulting pieces were suspended in 5 ml of HBSS, containing 2,500 U of collagenase Type 2 (Worthington Biochemical Corporation, Lakewood, NJ, http://www.worthington-biochem.com) and digested with shaking at 37¡ãC for 30 min. The digested sample was washed three times with cold HBSS. Islet purification was performed by centrifugation at 800g for 15 min at 4¡ãC on a discontinuous Ficoll (Sigma, St. Louis, http://www.sigma-aldrich.com) (1.08 mg/ml)/HBSS gradient. Islets were aspirated from the Ficoll/HBSS interface and washed with cold HBSS. Islet cell viability was confirmed by trypan blue exclusion.
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Dithizone Staining

Dithizone (DTZ) (Merck & Co., Whitehouse Station, NY, http://www.merck.com) stock solution was prepared as previously reported  by dissolving 50 mg of DTZ in 5 ml of dimethyl sulfoxide, was sterile-filtered through a 0.2 µm nylon filter, and was stored at ¨C20¡ãC. In vitro DTZ staining was performed by adding 10 µl of stock solution to 1 ml of culture medium. The culture dishes were then incubated at 37¡ãC for 30 min in DTZ-containing medium. After the dishes were rinsed three times with HBSS, crimson-red-stained clusters were examined with a stereomicroscope, and the number of DTZ-stained cells in cultures were determined.) W4 `' ]& t6 ]/ N- y

Fluorescence Labeling with Newport Green DCF9 Z1 l* H% P# _  P2 i: L, Z' m

The ester form of Newport Green (Molecular Probes Europe; Leiden, The Netherlands, http://probes.invitrogen.com) (diacetate form, NG-Ac) is cell-permeable. Inside the cell, this ester is cleaved by esterases to yield a cell-impermeant fluorescent indicator able to bind zinc ions. Cells in HBSS were exposed for 30 min at 37¡ãC to 25 mM NG-Ac and 1.5 µl/ml Pluronic F127 (Molecular Probes) to enhance the penetration of the probe. After being washed in HBSS, the cells were analyzed by confocal microscopy.

Immunocytochemistry

Immunocytochemistry was performed to detect the expression of insulin and other pancreas-specific hormones in differentiated cells. Cells cultured in Lab-Tek chambered slides (Nunc International, Rochester, NY, http://www.nuncbrand.com) were fixed with 4% paraformaldehyde in phosphate-buffered saline processed for immunofluorescence microscopy. The primary antibodies used for analysis were rabbit anti-insulin (sc-9168, Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), goat anti-glucagon (C-18) (sc-7779 Santa Cruz Biotechnology), rabbit anti-somatostatin (FL-116) (catalog number sc-20999 Santa Cruz Biotechnology), and goat anti-rat-C-peptide (Linco Research, catalog number 4023-01), goat monoclonal anti-insulin A (C-12), (catalog number sc-7839 Santa Cruz Biotechnology), rabbit anti-Ngn3 (kind gift from Prof. Michael S. German, UCSF Diabetes Center). The following secondary antibodies were used according to the manufacturer's recommendations: anti-goat IgG (whole molecule-Cy3 conjugate (catalog number C 2821, Sigma), anti-rabbit IgG (whole molecule)-Cy3 conjugated (catalog number C2306, Sigma), donkey anti-rabbit IgG (H L) labeled with Alexa Fluor 488 (catalog number A21206  , Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com), and donkey anti-goat IgG-rhodamine (sc-2094, Santa Cruz Biotechnology). Goat anti-rabbit IgG, Fc fragment-specific (Jackson ImmunoResearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com, code number 111-001-008), and rabbit anti-goat IgG (Nordic Immunological Laboratories, The Netherlands, http://www.nordiclabs.nl/index.htm) were used as isotype controls. Paraffin sections of adult mouse pancreas were taken as positive controls for the pancreatic hormones insulin, glucagon, and somatostatin. Paraffin sections from 16.5-day mouse embryos were taken as positive controls for Ngn3. Stained cells were analyzed by using confocal laser-scanning microscope (LSM META510 confocal scanning laser system consisting of an Axiovert 200 M microscope).
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Glucose-Regulated Insulin Secretion: V0 U! L% M3 V4 b
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To estimate secreted insulin levels, differentiated bone marrow cells were preincubated for 3 hours at 37¡ãC in Krebs Ringer-bicarbonate Hepes (KRBH) buffer containing 118 mM sodium chloride, 4.7 mM potassium chloride, 1.1 mM potassium dihydrogen phosphate, 25 mM sodium hydrogen carbonate, 3.4 mM calcium chloride, 2.5 mM magnesium sulfate, 10 mM Hepes, and 2 mg/ml bovine serum albumin (BSA) supplemented with 3.8 mM glucose. For high-glucose-induced insulin release, cells were further incubated in KRBH buffer supplemented with 7, 12, and 27.7 mM glucose, and 10 µM tolbutamide (Sigma) for 2 hours at 37¡ãC. The control was incubated with 3.8 mM glucose. Determination of secreted insulin was performed by using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Mercodia, Uppsala, Sweden, http://www.mercodia.com), according to the manufacturer's instructions. Statistical analysis was performed by Student's t-test." O4 ~, x7 S6 Q, J7 D  Y! t
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Immunoprecipitation and Western Blotting  `* m" _$ l$ `( L' M

Differentiated cells were assayed for the presence of intracellular insulin. The intracellular insulin was detected by cell extraction with lysis buffer and a combination of immunoprecipitation and Western blotting as described by Yang et al. . Specifically, the presence of insulin in the differentiated cell lysate was determined by immunoprecipitation with the rabbit polyclonal anti-insulin antibody (sc-9168), followed by separation on 18% SDS-polyacrylamide gel, transfer to nylon membranes, and blotting with anti-insulin antibody. Cell lysate (200 µg) was subjected to immunoprecipitation. Insulin protein was visualized by chemiluminescence.5 o; y& J4 M" u
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RNA Extraction and Reverse Transcription-Polymerase Chain Reaction Analysis7 H3 y- s' q8 J) i6 i) v

Total cellular RNA was extracted from whole bone marrow and differentiated cells by using TRIzol (Gibco/BRL), and reverse transcription was performed with 12¨C18 primer oligo(dT) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). cDNA samples were subjected to polymerase chain reaction (PCR) amplification with FastStart Taq DNA Polymerase (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) in 1.5 mmol/l magnesium chloride and 0.2 mmol/l dNTPs. The following transcription factors involved in endoderm development: PDX-1, Pax-4, and Ngn-3. Pancreas-specific genes, such as islet amyloid polypeptide, isl-1, insulin gene I and II, and glucose transporter two (GLUT-2) were analyzed by PCR. The cycling parameters were as follows: denaturation at 94¡ãC for 4 min, annealing at 50oC¨C64¡ãC for 1 min (depending on the primer), and elongation at 72¡ãC for 1 min (35 cycles). (Primers are listed in Table 1). PCR products of insulin gene I, insulin gene II, and PDX-1 were sequenced.
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Table 1. Primers for polymerase chain reaction

Electron Microscopy2 k$ m, C' s9 Z/ w, x. U
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For the postembedding detection of insulin in differentiated cells, samples were fixed with 0.2% glutaraldehyde and 0.5% formaldehyde for 1 hour on ice, washed with PBS containing 10 mM glycine, and dehydrated with a graded series of ethanol following the progressive lowering of temperature protocol (PLT method). Samples were then embedded in Lowicryl K4M (catalog number 15,923, Polysciences, Inc., Warrington, PA, http://www.polysciences.com) at ¨C35¡ãC, polymerized with UV light (366 nm, 2 days at ¨C35¡ãC, 2 days at room temperature), and ultrathin sections were cut with glass knives and collected onto formvar-coated copper grids (300 mesh). Sections were incubated with rabbit polyclonal anti-insulin antibody (sc-9168), (100 µg IgG protein/ml) overnight at 4¡ãC, washed with PBS, and incubated with gold markers (protein A/G gold, 15 nm in diameter from Biocell Cardiff, U.K., http://www.biocell.com) for 30 min at room temperature. After being washed with PBS containing 0.01% Triton X-100 and then with distilled water, samples were air-dried. Counterstaining was done with 4% uranyl acetate for 1 min. Samples were then examined in a Zeiss transmission electron microscope EM910 (Zeiss, Oberkochen, Germany, http://www.zeiss.com) at an acceleration voltage of 80 kV.: J/ E3 b. c& p
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RESULTS$ L- x% q8 S) y! c1 Z# L
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Chromatin-Remodeling Factors Induce Bone Marrow Cells to Differentiate into Islet-LikeCell Clusters
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To evaluate the effect of chromatin-remodeling factor TSA in the differentiation of BMSC into insulin-producing cells, murine bone marrow cells were cultured in the presence of TSA for 3 days and then maintained for an additional 7 days in differentiation induction medium containing 10 nM GLP-1 and high (25 mM) glucose. After removal of TSA, cells cultured under high-glucose medium started forming spheroid-like cell clusters from day 5 and attained their maximum size and maximal number at day 10. Bone marrow cells treated with TSA in the presence of high glucose formed islet-like clusters; this was not seen in control bone marrow cells cultured in the absence of TSA and high glucose (Fig. 1A). Cells cultured in the presence of TSA for 3 days had normal cell morphology (Fig. 1B). The three-dimensional cellular morphology of the cell clusters after 10 days culturing resembled pancreatic islet-like clusters (Fig. 1C, 1D), as described . The number of clusters formed was 300¨C350 per well in 6-well culture plates. The observed cellular morphology showed that bone marrow cells could differentiate into islet-like cells in the presence of TSA followed by culture in high-glucose medium.

Figure 1. Chromatin-remodeling factors induce bone marrow cells to differentiate into islet-like cell clusters. Bone marrow (BM) cells were treated in serum-free medium for 3 days with histone deacetylase inhibitor trichostatin A (TSA) and then cultured in 10% fetal bovine serum, glucagon-like peptide-1, and high glucose (25 mM) for an additional 7 days. BM cells were cultured for 10 days without any specific treatment (A). BM cells were cultured for 3 days in the presence of TSA (B). BM cells cultured for 10 days (3 7), 3 days in presence of TSA and subsequently cultured for an additional 7 days in high-glucose medium formed islet like clusters (x10) (C). At higher magnification, a single islet-like cluster appears to have defined edges and structure (x40) (D). Scale bar for (A¨CC): 100 µm and (D): 50 µm.: ]6 S8 X4 T4 _( B9 X" j( k. F, m1 I

Detection of Insulin-Containing Cells in Islet-Like Clusters by Using Zinc-Chelating Dyes1 g: G" H* ]" [3 O' r3 B8 C* F

To evaluate the insulin-producing cells in cultures, we stained the differentiated cells with the zinc-chelating agent DTZ and zinc-dependent fluorescence indicator Newport green (NG-Ac). Zinc is an integral part of insulin crystals forming the 2-Zn-insulin hexamer and is required by pancreatic ß-cells for packaging insulin. Free ionized zinc is present in the extragranular space as a reservoir for granular zinc. We took advantage of these zinc pools to identify cells harboring insulin synthesis in our cultures. DTZ is a zinc-binding substance, and pancreatic islets are known to stain crimson red after DTZ treatment. We first determined whether the isolated pancreatic islets from mouse were stained with DTZ and found that most islet cells were stained crimson red (data not shown). Differentiated individual cells and, in particular, cells in the islet-like clusters were distinctly stained crimson red by DTZ (Fig. 2A). Undifferentiated BMSC were not stained by DTZ (Fig. 2B).0 V$ Q7 h$ E! `7 k$ D$ S8 h. n% S
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Figure 2. Detection of insulin-containing cells in islet-like clusters. Insulin-containing cells in islet-like clusters were detected by using zinc-chelating agents, dithizone (DTZ), and Newport green (NG-Ac). On day 10, cells distinctly stained crimson-red by DTZ are apparent in the differentiated islet-like clusters (A). Undifferentiated BM cells are not stained (B). Individual differentiated cells stained with NG-Ac, showing green fluorescence within cytoplasmic dots (C). Control differentiated cells for NG-Ac stain (D). Scale bar for (A, B): 20 µm and for (C, D): 10 µm.
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Newport Green (NG-Ac) ester stains living cells at enzymatic cleavage by cellular esterases and subsequent binding to Zn2 . Confocal images of differentiated cells stained with NG-Ac exhibited green fluorescence dots (Fig. 2C). Staining of individual cells revealed a heterogeneous intensity of fluorescence with cytoplasmic dots. The control culture did not show significant staining with NG-Ac (Fig. 2D). No nuclear staining was noted. The presence of positive cells for zinc-specific dyes such as DTZ and NG-Ac suggest that insulin-producing cells can be derived from BMSCs.7 U3 P) r2 W* A% ~

Immunofluorescence Analysis for the Detection of Pancreas-Specific Hormones1 v8 h6 X; }2 ]9 I7 {2 b

To investigate the expression of pancreatic hormones, immunofluorescence analysis was performed for insulin, C-peptide, glucagon, and somatostatin in differentiated cells. Immunostaining of individual islet-like clusters, which were formed in BMSC cultures treated with TSA and subsequently cultured under high-glucose conditions, revealed large numbers of insulin-positive cells. Double-immunofluorescence analysis showed insulin (Fig. 3A, left), C-peptide (Fig. 3A, middle), and colabeling (Fig. 3A, right) in the same cell of islet-like clusters. Cells were counterstained by 4',6-diamidino-2-phenylindole to reveal the nucleus (Fig. 3A). The colocalization of C-peptide demonstrated de novo insulin synthesis and excluded the possibility that cells only absorbed and concentrated insulin from the medium. Glucagon- and somatostatin-expressing cells were also present in the culture. The staining for glucagon was found in the periphery of the cell (Fig. 3B), whereas somatostatin was dispersed in the cytoplasm (Fig. 3C). Costaining for insulin (Fig. 3D, left) and somatostatin (Fig. 3D, middle) showed that somatostatin-expressing cells were fewer in number than insulin-expressing cells in culture (Fig. 3D, right). Transcription factor Ngn3 was distinctly present in the nucleus of differentiated islet-like cells (Fig. 3E, middle and right). Matched isotype controls for rabbit IgG (Fig. 3F) and goat IgG (Fig. 3G) antibodies and without primary antibody (Fig. 3H) served as negative controls. These results indicate that (a) differentiated BMSC synthesize insulin de novo as indicated by the presence of C-peptide, (b) other pancreatic hormone-producing cells are also present in the culture after 10 days, and (c) the transcription factor Ngn3 is expressed by the islet-like cells.

Figure 3. Immunofluorescence analysis for detection of pancreas-specific hormones. Confocal microscopy of immunoassaying for insulin, C-peptide, glucagon, and somatostatin on day 10 islet-like clusters. Double-immunofluorescence analysis revealed insulin (green) (A), C-peptide (red), nuclear staining with DAPI (cyan); merging of both channels green (insulin) and red (C-peptide) was seen as yellow. Glucagon-positive cells (red) (B) and somatostatin-positive cells (red) (C) are present in the cultures. Costaining of insulin in green and somatostatin (D) in red was observed. Transcription factor Ngn3 was present in the nucleus of differentiated cells (E). In the isotype control for rabbit IgG (Isotype 1 ), no immunostaining is observed; all were counterstained by using DAPI (cyan) for nuclear staining. Scale bar 10 µm. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; Ngn3, neurogenin 3.+ G" u- S" H# u( ^0 b
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Adult mouse pancreas was used as a positive control for the hormones insulin, C-peptide, glucagon, and somatostatin (see supplemental materials). The transcription factor Ngn-3 was only detected at 16.5 days in mouse embryos (see supplemental materials), also as a positive control.% C( y8 F; \$ H" X

Protein Analysis of Islet-Like Clusters1 `' t& Q* ]/ A6 g! c$ S
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To determine whether the differentiated BM cells were responsive to glucose challenge, insulin release upon exposure to high glucose was measured by using an ultrasensitive mouse insulin ELISA. To enhance the sensitivity of these cells to the high-glucose challenge, the differentiated cells were switched to KRBH buffer containing 0.5% BSA and incubated in the presence of 3.8 mM glucose for 3 hours, then stimulated by the addition of 7, 12, or 27.7 mM glucose to the medium for 2 hours in individual experiments. Bone marrow-derived islet-like cells, after exposure to high glucose, secreted insulin in a glucose concentration-dependent manner (Fig. 4A). These data demonstrate that islet-like cell clusters derived from BMSC can secrete insulin in a glucose-regulated manner under appropriate conditions./ G0 D) s+ H5 M9 `

Figure 4. Protein analysis of bone marrow derived islet-like clusters. Analysis of secretion of insulin following high glucose challenge of differentiated BM-derived islet-like cultures. (A): Enzyme-linked immunosorbent assay for insulin in Krebs Ringer-bicarbonate Hepes (KRBH) buffer from BM-derived islet-like clusters exposed to low- (3.8 mM) and high-glucose conditions 7, 12, and 27 mM for 2 hours. After exposure to high glucose, BM-derived islet-like clusters secreted insulin. Statistical significance tested by Student's t-test: *, p
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To analyze the synthesized and stored insulin in differentiated clusters, cell lysates of day 3 culture and day 10 cultures after TSA treatment were subjected to immunoprecipitation and Western blot analysis. Cell lysates of differentiated bone marrow cells contained stored insulin. The cell lysate of day 3 cultures contained an immunopositive band, and the band intensity increased in cell lysate at day 10 (Fig. 4B). Pancreatic tissue served as a positive control. In contrast, no corresponding band was detectable in untreated BMSC (Fig. 4B). The data of insulin protein analysis suggest that the differentiated BMSC in islet-like clusters synthesize and store insulin.
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Endocrine-Specific Gene Expression in Differentiated Islet-Like Clusters' ~! @/ z1 q) E5 Z1 B3 l

Pancreatic development and gene expression are regulated by specialized transcription factors. To determine whether endocrine-specific transcription factors and pancreas-specific genes were expressed during the differentiation into islet-like clusters, reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed at different time points. Transcripts for PDX-1, Pax 4, HNF-3ß, and Isl-1 were not detectable in undifferentiated BMSC; in contrast, they were upregulated in differentiated cells (Fig. 5). PDX-1 and HNF-3ß transcripts were expressed on day 3 after TSA treatment and increased by day 10. The PCR product of neurogenin 3 (Ngn-3) was visible in both undifferentiated and differentiated BMSCs. PCR products for insulin gene I and II were visible at day 3 and day 10. In contrast, other major islet-specific hormones, such as IAPP, glucagon, and somatostatin, were not detected on day 3 of differentiation and were expressed only on day 10. PCR products for the ATP-sensitive potassium channel-specific sulfonylurea receptor (SUR1) and glucose transporter gene (GLUT-2) were detectable only at day 10. Gene expression analysis in BMSC derived islet-like clusters was similar to that in the pancreas tissue and confirmed the differentiation of BMSC into islet-like cells in vitro upon treatment with TSA and subsequent culturing in high glucose.
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Figure 5. Endocrine-specific gene expression in differentiated islet-like clusters. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of pancreas-specific gene expression at several differentiation stages. Total RNA isolated from both undifferentiated and differentiated bone marrow cells was subjected to RT-PCR analysis with primers for the indicated genes. Lane BM: undifferentiated whole bone marrow. Lane D3: day 3 culture with trichostatin A only. Lane D10: day 10 culture with high glucose and glucagon-like peptide-1. Lane Pancreas: adult mouse pancreas (positive control). Lane -ve RT: No template (negative control). Abbreviations: BM, bone marrow; D, day.7 u0 ?6 u6 G) k; h; _, s; W% q
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Ultrastructural Analysis of Insulin-ProducingCell Clusters0 O" Q6 C( Y2 F0 E3 M# W5 h% p1 _
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Ultrastructural analysis of bone marrow derived insulin-producing clusters was also performed. Differentiated cells at low magnification revealed a structure typical of a secretory cell, with secretory vesicles containing dense granules (Fig. 6A). Immunogold electron microscopy showed insulin within the small secretary vesicles of the insulin-producing clusters (Fig. 6B). Gold-labeling detected faint globular structures of differing size filled with low-density material. Positive and negative controls for immunogold labeling are presented in Figure 6C and 6D, respectively. Adult pancreatic ß-cells showed several positive immunogold particles per secretory granule (Fig. 6C). The pancreatic ß-cells were examined only to validate the assay and not to correlate insulin-positive signals between the ß-cells and the BM-derived insulin-producing clusters. Control bone marrow cells showed no specific labeling, but a few nonspecific particles were seen in the cytoplasm (Fig. 6D). The ultrastructural study of differentiated bone marrow cells showed features typical of an adult ß-cell, and insulin granules were observed within the secretory vesicles suggesting that these cells had differentiated into pancreatic ß-like cells capable of synthesizing insulin.3 s$ E8 J  P* M

Figure 6. Ultrastructural analysis of insulin-producing cell clusters. Postembedding immunogold staining for insulin in day 10 islet-like clusters. Secretory granules (arrow) are densely packed within the cytoplasm of the differentiated cell (A). At higher magnification, insulin granules (arrows) are seen in secretory vesicles (B). (C): Positive control for immunogold labeling: positive granules (arrows) in adult mouse pancreas. (D): Negative control. Abbreviation: N, nucleus./ [/ A0 q$ Q2 n
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DISCUSSION7 }* \  x! V& U4 G

Stem cells are a potential source for ß-cell replacement therapy. Fundamental processes in the determination of the differentiation pathways of stem cells remain to be elucidated and robust and reliable differentiation protocols need to be established. ES cells can be differentiated into insulin-producing cells by manipulating culture conditions .

ES cells contain potent chromatin-remodeling activities; observations suggest that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells. For a better understanding of stem cell differentiation, chromatin dynamics should be considered. The reprogramming of bone marrow cells to insulin-producing cells may depend on chromatin modulation. To evaluate whether chromatin reprogramming can contribute to the in vitro differentiation of BM cells into insulin-producing cells, we have added the histone deacetylase inhibitor (HDACi), TSA, to the culture medium. TSA is the best-studied HDACi .6 r1 x: O, x4 S1 p, g4 k; @1 c) N" R

We have explored the possibility of using mouse BM cells as a source for insulin-producing cells following treatment with TSA and subsequently culturing in the presence of high glucose and GLP-1. Glucose is well known as a growth factor for ß-cells . We have generated functional insulin-producing cells from bone marrow and confirmed the presence of insulin production by RT-PCR, immunofluorescence, Western blot, and electron microscopy combined with immunogold anti-insulin labeling. Furthermore, we have tested the functionality of the in vitro generated insulin-producing cells from BM by measuring insulin release in response to high glucose concentrations. Taken collectively, these studies provide evidence that the BM contains pluripotent cells capable of being reprogrammed in vitro by TSA to become functional insulin-producing cells.

In addition, we have found transcripts for proinsulin I and II by RT-PCR. Transcripts for the component of the ATP-sensitive K  channel SUR1 and for GLUT-2, which participates in the signal-mediated secretion of insulin in pancreatic ß-cells, have also been detected. Moreover, transcription factors Isl 1, Pax 4, Ngn-3, and IAPP are present at later stages of differentiation. Pax4 is required for the development of cells restricted to the ß- and -cell lineages. Mice lacking Pax4 fail to develop any ß-cells and become diabetic .

We have also detected the expression of glucagon and somatostatin by PCR, and the respective proteins have been detected by immunocytochemistry. We have found fewer somatostatin-positive and glucagon-positive cells in culture by single-staining immunocytochemistry in contrast to the large number of insulin-positive cells in culture. Double-staining for insulin and somatostatin has confirmed the low numbers of somatostatin-containing cells.

PDX-1 gene expression is low at earlier stages of differentiation; PDX-1 expression at these stages may initiate a cascade of events leading to insulin transcription. Although at later stages, high levels of PDX-1 transcript are found, we have failed to detect PDX-1 by immunocytochemistry. This is not surprising, because it is known that when the mRNA for PDX-1 is highly abundant, immunoreactivity can hardly be detected .6 u4 ?+ K- M$ v! p% x5 W3 |4 }

During embryogenesis, only cells expressing Ngn-3 are islet progenitors  have shown that cells coexpressing glucagon, insulin, and Ngn-3 eventually become mature ß-cells. The islet-like clusters in our culture system express insulin, glucagon, and Ngn-3 and thus strongly resemble pancreatic precursor cells. This has been confirmed by our immunocytochemical results.7 y6 O) F7 s: w9 g' ^0 L
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The C-peptide expression in islet-like cells and glucose-dependent insulin release provide evidence that these cells synthesize and release insulin. C-peptide is a by-product of insulin synthesis, and therefore the demonstration of C-peptide and the colocalization of insulin are reliable methods to investigate pancreatic differentiation in vitro. In our study, BM cell differentiate into cells coexpressing insulin and C-peptide; this rules out the uptake of insulin from the culture medium, as mentioned above. The data confirm that BM cells are capable of differentiating into pancreatic cells producing insulin de novo. This secretion of insulin seems to be dependent upon the concentration of glucose in the medium; however, osmotic effects cannot be ruled out, although Lumelsky et al.  have found no such effects with sucrose.

In summary, we have shown here that murine BM cells are reprogrammed into insulin-producing cells by treatment with the histone deacetylase inhibition factor, TSA, and following culture in a high-glucose medium. However, further research has to be carried out on chromatin-remodeling factors to understand the mechanisms that are involved in cell-fate determination. The insulin-producing cells secrete a substantial amount of insulin, and immunostaining and RT-PCR analysis have revealed that islet-like cells derived from bone marrow are similar to pancreatic cells. The development of new protocols to differentiate BM to pancreatic ß-like cells may enable the use of these cells for future cell therapy of type 1 diabetes.
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DISCLOSURES$ Q6 b9 W# s( E. e* r
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS8 l; l3 h: d( A
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The work was supported by a Grant MA 852/7-3 from Deutsche Forschungsgemeinschaft (DFG). We thank P. Paul M¨¹ller and Dr. Theresa Jones for critically reading the manuscript and Prof. Michael S. German UCSF Diabetes Center, CA for providing aliquots of Ngn3 antibody.& i9 D6 R' B' A- `% R. _+ _' F
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