Role of BMP Signaling in Pancreatic Progenitor Differentiation from Human Embryonic Stem Cells

Transplantation of pancreatic progenitors derived from human embryonic stem cells (hESCs) is a promising way to treat diabetes. Strategies to obtain the required cell mass would rely on the up scaling of current differentiation protocols, or the proliferation of committed progenitors. We aimed at finding conditions that maintain a proliferating pancreatic progenitor pool and we assessed the role of BMP4 signaling in this process. hESCs were differentiated into PDX1 positive pancreatic progenitor stage following our established protocol with few modifications, and then the progenitor cells were passaged in a defined proliferation medium (PM). During passage, the effect of BMP4 signaling on the differentiation and proliferation of pancreatic progenitors was examined by RT-PCR and immunofluorescence analysis. We found that PDX1 positive pancreatic progenitors proliferated and gained NKX6.1 expression in the PM, whereas they failed to express NKX6.1 if BMP signaling was inhibited with Noggin. In this latter condition, part of the progenitors rather generated pro-endocrine cells denoted by NGN3 and synaptophysin expression. On the contrary, addition of BMP4 to the PM promoted the early derivation of PDX1 and NKX6.1 coexpressing pancreatic progenitors. Our findings are in line with mouse pancreas development, and indicate that BMP4 signaling is required for the derivation and maintenance of hESC-derived PDX1+NKX6.1+ pancreatic progenitors. These results are instructive for guiding the development of an efficient pancreas differentiation protocol in view of diabetes cell replacement therapy.     

神经前体细胞不是胚胎干细胞向胰岛素分泌细胞分化的必经途径

探讨胚胎干细胞向胰岛素分泌细胞分化的途径,对胰腺组织工程的临床运用有重要意义。将胚胎干细胞在有小鼠胚胎成纤维细胞饲养层和白血病抑制因子的条件下培养扩增后,再将扩增后的胚胎干细胞不经过神经前体细胞阶段直接诱导为胰岛素分泌细胞,并与传统的多阶段诱导(经过神经前体细胞阶段)进行比较。结果发现,胚胎干细胞脱离饲养层细胞后,经过9~10d的分化诱导,可以分化为具有胰岛β-细胞特征的胰岛素分泌细胞。与传统的多阶段诱导方法相比,诱导过程简化,诱导时间缩短,所得到的胰岛素分泌细胞数量无明显差异。说明胚胎干细胞向胰岛素分泌细胞分化存在多条途径。神经前体细胞阶段不是胚胎干细胞向胰岛素分泌细胞分化的必须途径。用传统的多阶段分化诱导法和直接诱导法都可以将胚胎干细胞诱导成胰岛素分泌细胞。     

小鼠骨髓间充质干细胞向胰岛素分泌细胞诱导分化过程中调控PDX-1基因表达miRNAs的鉴定

目的实现小鼠骨髓间充质干细胞(BMSCs)向胰岛素分泌细胞(IPCs)的诱导分化并对分化过程中可能调控胰十二指肠同源异型盒基因-1(PDX-1)基因表达miRNAs进行鉴定。方法首先分离培养BMSCs,应用conophylline和尼克酰胺将其诱导分化为IPCs,采用双硫腙(DTZ)染色和免疫荧光检测胰岛素的表达。然后采用靶基因预测软件miRanda和Target Scan对调控PDX-1基因表达miRNAs进行预测并通过双荧光素酶报告基因系统鉴定。实时定量聚合酶链反应(Real-time PCR)检测诱导分化过程中miRNAs及PDX-1的表达。结果诱导分化后的细胞双硫腙染色呈猩红色,免疫荧光化学显示有胰岛素表达。生物信息学方法预测得到4个可能调控PDX-1表达的miRNAs,通过双荧光素酶报告基因系统检测发现其中的miR-149和miR-346能结合到PDX-1 mRNA的3’UTR并有效抑制其表达。Real-time PCR检测结果表明,miR-149和miR-346的表达水平与PDX-1表达呈负相关。结论 miR-149和miR-346能负性调控IPCs诱导分化过程中PDX-1的表达。     

Use of long-term cultured embryoid bodies may enhance cardiomyocyte differentiation by BMP2

Purpose

Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs.

Materials and Methods

hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed.

Results

Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and α-myosin heavy chain (αMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and α-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS.

Conclusion

We demonstrated that the use of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.     

EphB3 marks delaminating endocrine progenitor cells in the developing pancreas

Background: Understanding the process by which pancreatic beta‐cells acquire their “fate” is critical to the development of in vitro directed differentiation protocols for cell replacement therapies for diabetics. To date, these efforts are hampered by a paucity of markers that distinguish pancreatic endocrine cells at different stages of differentiation. Results: Here, we identify EphB3 as a novel pro‐endocrine marker and use its expression to track delaminating islet lineages. First, we provide a detailed developmental expression profile for EphB3 and other EphB family members in the embryonic pancreas. We demonstrate that EphB3 transiently marks endocrine cells as they delaminate from the pancreatic epithelium, prior to their differentiation. Using a Tet‐inducible EphB3^rtTA‐lacZ reporter line, we show that short‐term pulse‐labeled EphB3⁺ cells co‐express Pdx1, Nkx6.1, Ngn3, and Synaptophysin, but not insulin, glucagon, or other endocrine hormones. Prolonged labeling tracks EphB3⁺ cells from their exit from the epithelium to their differentiation. Conclusions: These studies demonstrate that pro‐endocrine cell differentiation during late gestation, from delamination to maturation, takes approximately 2 days. Together, these data introduce EphB3 as a new biomarker to identify beta‐cells at a critical step during their step‐wise differentiation and define the timeframe of endocrine differentiation. Developmental Dynamics 241:1008–1019, 2012. © 2012 Wiley Periodicals, Inc.     

β-Cell neogenesis: experimental considerations in adult stem cell differentiation

The contribution of stem cells derived from adult tissues to the recovery of pancreatic islets from chemical injury is controversial. Analysis of nonhematopoietic differentiation of bone marrow-derived cells has yielded positive and negative results under different experimental conditions. Using the smallest subset of bone marrow cells lacking immuno-hematopoietic lineage markers, we have detected incorporation and conversion into insulin-producing cells. Donor cells identified by genomic markers silence green fluorescent protein (GFP) expression as a feature of differentiation, in parallel to expressing PDX-1 and proinsulin. Here we elaborate potential experimental difficulties that might result in false-negative results. The use of GFP as a reporter protein is suboptimal for differentiation experiments: (a) the bone marrow of GFP donors partially expresses the reporter protein, (b) differentiating bone marrow cells silence GFP expression, and (c) the endocrine pancreas is constitutively negative for GFP. In addition, design of the experiments, data analysis, and interpretation encounter numerous objective and subjective difficulties. Rigorous evaluation under optimized experimental conditions confirms the capacity of adult bone marrow-derived stem cells to adopt endocrine developmental traits, and demonstrates that GFP downregulation and silencing is a feature of differentiation.     

小鼠iPSCs诱导分化为胰岛素分泌细胞及治疗糖尿病的实验研究

 目的: 利用3步法诱导方案,使小鼠诱导多能干细胞(induced pluripotent stem cells,iPSCs)分化为胰岛素分泌细胞(insulin-producing cells,IPCs),观察诱导效率和成熟度,并观察其对糖尿病小鼠治疗效果。方法: 本实验室通过piggyBac转座子将C57/C雄性小鼠来源胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)构建为小鼠iPSCs,利用3步法诱导方案分化为IPCs,观察细胞分化过程中的形态变化;RT-PCR和免疫荧光检测其胰岛β细胞发育相关基因和蛋白的表达;流式细胞术分析分化效率;葡萄糖刺激实验检测胰岛素和C肽分泌水平。将IPCs移植入C57/C雄性糖尿病小鼠模型左肾包膜下,监测血糖和血清中胰岛素含量28 d,观察逆转高糖血症的效果。结果: 建立的3步诱导方案可以将小鼠iPSCs诱导分化为IPCs,其表达胰岛β细胞的标志性基因(Pdx1、Ngn3、Pax6和Ins2)和蛋白(Pdx1、Nkx6.1和胰岛素),在葡萄糖刺激下可分泌胰岛素和C肽。流式细胞术结果表明诱导分化的效率达28%。移植后3 d糖尿病小鼠血糖即降低至接近正常水平,血清胰岛素含量明显升高,对葡萄糖的调控能力明显增强。病理学观察IPCs细胞移植28 d后仍存活。结论: 建立的3步诱导法可将iPSCs高效定向诱导分化为IPCs,明显缩短了诱导分化的时间,移植至近交系同性别小鼠体内可逆转糖尿病的高糖血症。     

Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development

Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mRNA and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. Disclosure of potential conflicts of interest is found at the end of this article.     

Presenilins,Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window

Canonical Notch signaling is thought to control the endocrine/exocrine decision in early pancreatic progenitors. Later, RBP-Jκ interacts with Ptf1a and E12 to promote acinar differentiation. To examine the involvement of Notch signaling in selecting specific endocrine lineages, we deregulated this pathway by targeted deletion of presenilin1 and presenilin2, the catalytic core of γ-secretase, in Ngn3- or Pax6-expressing endocrine progenitors. Surprisingly, whereas Pax6⁺ progenitors were irreversibly committed to the endocrine fate, we discovered that Ngn3⁺ progenitors were bipotential in vivo and in vitro. When presenilin amounts are limiting, Ngn3⁺ progenitors default to an acinar fate; subsequently, they expand rapidly to form the bulk of the exocrine pancreas. γ-Secretase inhibitors confirmed that enzymatic activity was required to block acinar fate selection by Ngn3 progenitors. Genetic interactions identified Notch2 as the substrate, and suggest that γ-secretase and Notch2 act in a noncanonical titration mechanism to sequester RBP-Jκ away from Ptf1a, thus securing selection of the endocrine fate by Ngn3 progenitors. These results revise the current view of pancreatic cell fate hierarchy, establish that Ngn3 is not in itself sufficient to commit cells to the endocrine fate in the presence of Ptf1a, reveal a noncanonical action for Notch2 protein in endocrine cell fate selection, and demonstrate that acquisition of an endocrine fate by Ngn3⁺ progenitors is γ-secretase-dependent until Pax6 expression begins.     

Insulin and Glucagon mRNA Expression and Prohormone Convertase Immunoreactivity in Normal and Neoplastic Pancreatic Endocrine Tissue

Prohormone convertase (PC) 1/3 and PC2 are neuroendocrine-specific enzymes that convert prohormones to active hormones. To learn more about the role of these PCs in normal and neoplastic islet cells, we analyzed a series of pancreatic endocrine tumors to determine the role of PC1/3 and PC2 in islet cell hormone processing. In situ hybridization with insulin and glucagon probes and immunostaining with antibodies to PC1/3 and PC2 were done with 6 normal pancreases, 33 insulinomas, and 7 glucagonomas. The intensity of the reactions was graded from 0 to 3+. Normal islets stained strongly for both proinsulin and proglucagon mRNAs. Insulinomas and glucagonomas had readily detected hormone mRNAs for proin sulin and proglucagon, respectively. Normal pancreatic insulin cells stained weakly for PC1/3 (1+) and PC2 (1−2+), and glucagon cells stained weakly for PC1/3 (1−2+) and strongly for PC2 (2−3+). Both insulinomas and glucagonomas stained strongly for PC2 (2−3+) with less intense staining for PC1 (1−2+). These results indicate that PC2 is more highly expressed in insulin- and glucagon-producing pancreatic islet cell tumors and that there is increased expression of PC2 in insulinomas compared to normal insulin-producing cells.