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a Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, China;
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b Institute of Biological Science and Technology, College of Science, Beijing Jiaotong University, Beijing, China Z. z6 ]. |5 V) ]( {4 p" K3 P
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Key Words. Embryonic stem cells ? Pancreatic ? cells ? Differentiation ? All-trans retinoic acid ? Activin A' b& p# ?- K2 I; ^8 l
6 P H' ~! Z3 e7 o* W7 uCorrespondence: Hongkui Deng, Ph.D., Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, 100871, P. R. China. Telephone: 86-10-6275-6954; Fax: 86-10-6275-6954; e-mail: hongkui_deng@pku.edu.cn; and Mingxiao Ding, M.S., Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, 100871, P. R. China. Telephone: 86-10-6275-6954; Fax: 86-10-6275-6954; e-mail: dingmx01@pku.edu.cn! V+ A2 W( L( |( U$ E/ }* g
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ABSTRACT
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) U8 E5 z! i/ W( V2 vDiabetes mellitus affects 4%–5% of the world’s population and is the most common metabolic disorder in humans. The number of people with diabetes is predicted to exceed 350 million by 2010. Type I diabetes mellitus results from the autoimmune destruction of the ? cells in pancreatic islets. Many research groups are therefore exploring ways to replace these destroyed insulin-producing cells. Until now, pancreatic islet cell transplantation is the only effective approach to cure type I diabetes instead of insulin injection . However, this therapy is not widely used because of the severe shortage of transplantable donor islets.* b U3 r. t; b7 H `# E
; S9 E& N& ?3 R. x) IOne attractive approach is the generation of functional ? cells from embryonic stem cells (ESCs). ESCs have been shown to be able to differentiate into pancreatic islet–like clusters, especially pancreatic ? cells . First, Soria et al. successfully induced ESCs to differentiate into pancreatic ? cells by a cell-trapping system. However, this is a complicated process with genetic manipulation. Then Lumelsky et al. designed a five-stage protocol to induce ESCs to differentiate into insulin-producing islet-like structures without genetic modification. Hori et al. and Blyszczuk et al. improved Lumelsky’s five-stage induction protocol by adding growth inhibitor LY294002 or overexpression of the pax4 gene. However, all these induction approaches are somewhat complicated and take a long period of time. Later Hansson et al. used the five-stage induction protocol and found that ESC-derived insulin-positive cells absorbed insulin from the culture medium instead of producing it themselves. Therefore, it is necessary to find the specific induction factors that can induce ESCs to differentiate into pancreatic ? cells more simply and rapidly.5 O* T' z4 K) t* E \
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It has been suggested that some factors are able to promote definitive endoderm differentiation. Activin A, a member of the transforming growth factor–beta (TGF-?) superfamily, is critical for mesoderm and endoderm formation during gastrulation. When used at a high concentration, it primarily induces endoderm formation . All-trans retinoic acid (RA) is a well-characterized signaling molecule that acts in anteroposterior patterning of neuroectoderm and mesoderm in vertebrates . Recent evidence indicates that RA is also involved in the regulation of the embryonic endoderm differentiation pattern, especially the early pancreas bud formation, and it is able to improve insulin expression in pancreatic ? cells and the INS-1 cell line . It has been demonstrated that the combination of activin A and RA was able to induce the Xenopus presumptive ectoderm region of the blastula to differentiate into pancreatic insulin-positive cells .
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3 |' p" R1 S7 P1 IWe report here a novel three-step approach based on combination of activin A, RA, and other factors that mature pancreatic ? cells. This three-step protocol can induce ESCs to differentiate into insulin-producing cells within only 2 weeks. These insulin-positive cells express characteristic pancreatic ?-cell marker genes such as insulinI, pdx1, glut2, hnf3?, and is11. Moreover, we use insulin promoter–enhanced green fluorescent protein (EGFP)–marked ESCs to further demonstrate that this strategy can indeed induce ESCs to differentiate into insulin-producing cells instead of uptaking insulin. Finally, we provide evidence that insulin-producing cells are able to fully rescue the streptozocin (STZ)–induced diabetic mice when they are transplanted under their renal capsules.
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. L9 c+ K. f+ W, q5 s2 aMATERIALS AND METHODS# v2 f9 ~, c; N2 f! }. f; ^" g7 w* D/ e
- {2 W! H; u% a+ h! n8 LInducing ESCs to Insulin-Positive Cells by Activin A and RA( l- U% E# Y! A( h/ R% |
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We designed a novel three-step induction strategy based on activin A and RA (Fig. 1A). First, ESCs (Fig. 1B) were suspended for EB formation. Then EBs were placed into 1% Matrigel-coated dishes and induced by activin A and RA sequentially. Second, the cells were switched to a medium containing 10% FBS/DMEM and bFGF, then cultured for another 3 days, which improved the proliferation of pancreatic progenitor cells derived from EBs. During this stage, most of the remaining cells were epithelial-like (Fig. 1C). Some new clusters appeared from large, spreading EBs. Third, the cells were cultured in DMEM/F12 medium with N2 supplement, B27 supplement, laminin, bFGF, and nicotinamide, which appeared to promote pancreatic ? cells to maturation . At this stage, many small cell clusters appeared (Fig. 1D, E).+ l1 C6 C, {# [4 C
6 A Z9 E3 M( h( L1 L& LFigure 1. Protocol of embryonic stem (ES) cell differentiation and cell morphology change during induction. (A): The three-step protocol induced ES cells to differentiate into insulin-positive cells. Cell morphology changed in different induction steps (x200). (B): In step 1, the ES cell R1 cluster was cultured in 20% fetal bovine serum/Dulbecco’s modified Eagle medium (FBS/DMEM) with 1,000 U/ml leukemia inhibitory factor. (C): In step 2, after activin A and retinoic acid (RA) interval induction, embryoid bodies spread, and most of the living cells were epithelial-like when cultured in 10% FBS/DMEM with 10 ng/ml basic fibroblast growth factor (bFGF). (D, E): In step 3, after culturing in DMEM/F12 with N2, B27, nicotinamide, bFGF, and laminin, the cells formed small cluster-like constructions.% p, A/ X, f; @
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To test whether these factors promote the ESCs to differentiate into pancreatic endocrine cells, we detected the expression of several marker genes in induced cells by RT-PCR. We found that the induced cells expressed many characteristic pancreatic ?-cell marker genes, such as pdx-1, insulinI, glut2, is11, and hnf3? at the third step of our protocol. The epithelial-like cells induced by activin A or RA alone expressed hnf3? or pdx-1 at the end of the second step (Fig. 2A). In the second step, the differentiated epithelial-like cells expressed pancreatic progenitor markers such as hnf3?, hnf4, and pdx1 (Fig. 2B). If induced by the three-step protocol with both activin A and RA, the differentiated cells strongly expressed pdx1, hnf3?, insulinI, glut2, and is11 (Fig. 2C, lane 3). These cells did not express markers of other islet endocrine cells (non-? cell) such as glucagon or somatostatin (data not shown). This result suggests that TGF-? and RA signalings may be specific for pancreatic ?-cell development and maturation. We found this induction was strictly dependent on activin A and RA, as absence of both of them resulted in almost no small cluster formation and the induced cells did not express or only weakly expressed pdx1, hnf3?, insulinI, glut2, and is11 (Fig. 2C, lane 2). Moreover, we found that if the dishes were coated with laminin or gelatin, instead of Matrigel, no cell clusters formed in the third step.
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Figure 2. Reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression of differentiated cells at the end of step 2 and step 3. (A): The top panel shows that hnf3? and pdx-1 were expressed when treated with activin A alone; the bottom panel shows that only hnf3? was expressed when treated with retinoic acid (RA) alone. (B): Gene expression of the cells after the induction in step 2. (C): Analysis of gene expression of cells induced by the three-step protocol. Lane 1 (pancreas), adult mouse pancreas sample as the positive control; lane 2 (– –), gene expression of cells induced by the three-step protocol without activin A and RA; lane 3 ( ), gene expression of cells induced by the three-step protocol with activin A and RA. Lane 4 (RT) is the same sample of lane 3, but PCR was performed without reverse transcriptase.
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% S2 p1 P5 F% W( t+ \) A2 {7 |To determine whether there were insulin-positive and C-peptide–positive cells, we performed immunohistochemistry staining to detect insulin and C-peptide expression in the induced cells. The results showed that ESC-derived cell clusters expressed both insulin and C-peptide (Fig. 3A, B). If cultured without activin A and RA, the cells did not express insulin (Fig. 3C, D). In addition, we also used DTZ to detect the induced cells and found that some clusters in the third step appeared crimson red after DTZ staining (data not shown). This proved that, within those clusters, there were cells that had the same characteristics as pancreatic ? cells.
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0 J" j& h/ l eFigure 3. Analysis of insulin expression and insulin release of induced cells. (A): The cells induced by the three-step protocol stained with primary antibody to insulin were insulin-positive. (B): The cells induced by the three-step protocol stained with primary antibody to C-peptide were C-peptide–positive. (C): The cells without activin A and retinoic acid induction did not express insulin (x200). (D): The cells induced by the three-step protocol stained with primary antibody to insulin were insulin positive (x200). (E, G): The cells derived from the enhanced green fluorescent protein (EGFP) reporter–marked embryonic stem cells induced by the three-step protocol were EGFP positive (x200). (F, H): Those EGFP cells stained by primary antibody to insulin were also insulin-positive (x200). (I): Insulin release detection by enzyme-linked immunosorbent assay. Insulin level after treatment with 27.7 mM glucose medium (column a) was nearly six times higher than the insulin level in the 5.5-mM glucose medium (column b).
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, }! }6 [' R+ F/ H$ \There is evidence showing that ESC progeny could take up the insulin from the culture medium and then stained positive by insulin antibody . To confirm whether the insulin was generated by the induced clusters, we also used an EGFP-reporter system to detect the insulin expression. We transfected the R1 ESCs with an EGFP-reporter vector, in which a green fluorescent protein cDNA was driven by an insulin promoter. ESCs that have incorporated this vector were selected by G418 and induced with activin A and RA. In the third step, EGFP expression was observed within the small cluster cells (Fig. 3E, G), and their expression pattern matched that of insulin detected by immunohistochemistry (Fig. 3F, H). These data indicated that activin A and RA, combining with other mature factors, could indeed induce ESCs to differentiate into insulin-producing cells.. z' X* q, y+ L) J$ X* k# f6 u* [8 F
0 h1 y7 e" ~, r) ~% _To analyze whether insulin secretion from those differentiated ESCs could be regulated by glucose, we treated about 106 induced cells with Krebs-Ringer buffer containing either low (5.5 mM) or high (27.7 mM) concentration of glucose and then analyzed the insulin release level in the culture medium by ELISA. We found that the insulin release in the high-glucose medium was nearly six times higher than that in the low-glucose medium (Fig. 3I). This result suggested that the ESC-derived insulin-positive cells secreted insulin in a glucose-dependent manner, as do normal pancreatic ? cells.5 z8 q8 ^3 |$ B
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Transplantation of ESC-Derived Insulin-Positive Cells& R6 F' n! f6 X/ _0 K+ A4 c4 ?
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To investigate whether the ESC-derived insulin-producing cells could rescue mice with diabetes, we transplanted the induced cells into the left renal capsules of STZ-treated diabetic mice (n = 9). After transplantation, the survival probability of cell-transplanted mice was about three times higher than that of PBS sham-operated control mice (n = 12) (Fig. 4A). The survival mice continued to gain weight. Two weeks later, the blood glucose of cell-treated mice reduced to a normal level (less than 13.9 mM). In contrast, the blood glucose of the PBS sham-operated control mice was still above 16 mM (Fig. 4B). In addition, after removal of the left kidney transplanted with ESC-derived cells, the blood glucose level of STZ-treated mice reversed back to over 13.9 mM within 3 days, and these mice could survive for nearly 1 week. The recrudescent diabetes and weak body may be the main reasons for their death. Cryostat sections of the operated kidneys were prepared, and insulin-positive cells could be visualized only in the differentiated cell–treated kidneys by immunohistochemistry (Fig. 5A, B). These results showed that the ESC-derived insulin-producing cells, when transplanted into the renal capsule, could improve glucose control and functionally rescue the STZ-treated mice with diabetes.
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: U. [3 Y. `+ W% j- ZFigure 4. Transplantation of embryonic stem cell–derived insulin-positive cells into diabetic mice left renal capsule ameliorates the diabetic symptom. (A): Differentiated cell–treated diabetic mice (n = 9, dashed line) survived longer, and the survival probability reached 70%, while the survival probability of phosphate-buffered solution (PBS)–transplanted diabetic mice (n = 12, solid line) was 25%. (B): Blood glucose level analysis of induced cell- (n = 5) and PBS- (n = 3) transplanted streptozocin-diabetic mice. Blood glucose levels of cell-transplanted diabetic mice recovered to normal (13.9 mM) (white circle).! r+ Q- ?8 I. E8 c& h6 I1 b5 Z
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Figure 5. Analysis of insulin expression in transplanted kidney of 129 diabetic mice. (A): Insulin-positive cells were not detected in phosphate-buffered solution–injected left kidney with primary antibody to insulin (x200). (B): Insulin-positive cells could be detected only in the differentiated cell–treated kidney of streptozocin-diabetic mice (x200).
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$ q) b x6 P# H# H) _ g+ z' RThis research was supported by grants from the Ministry of Science and Technology (no. 2001CB510106) and the Science and Technology Plan of Beijing Municipal Government (no. H020220050290); an award from the National Nature Science Foundation of China for Outstanding Young Scientists (no. 30125022) to H.D.; and a grant from the Ministry of Science and Technology (no. 1999053900) to M. D.
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We thank Dr. M.S. German for kindly providing the insulin promoter vector. We also thank Dr. Tung-Tien Sun for critical reading of the manuscript. In addition, we acknowledge other colleagues in our lab for advice during experiments. Y.S., L.H., and F.T. contributed equally to this article.
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发表于 2009-3-5 10:50
发表于 2015-5-29 17:43
