A secretory function of human insulin-producing cells in vivo

BACKGROUND:Mesenchymal stem cells derived from human umbilical cord blood(UCB-MSCs)have good research and application prospects in the treatment of diabetes.We once induced UCB-MSCs to differentiate into insulin-producing cells(IPCs)in vitro,but we did not know the functions of these cells in vivo.The aim of this study was to assess the functional effects of IPCs on insulin secretion and their role in the treatment of diabetes in vivo. METHODS:UCB-MSCs were induced to IPCs by an inducing protocol with extra...     

Generation of insulin-producing cells from pluripotent stem cells: from the selection of cell sources to the optimization of protocols

The pancreas arises from Pdx1-expressing progenitors in developing foregut endoderm in early embryo. Expression of Ngn3 and NeuroD1 commits the cells to form endocrine pancreas, and to differentiate into subsets of cells that constitute islets of Langerhans. β-cells in the islets transcribe gene-encoding insulin, and subsequently process and secrete insulin, in response to circulating glucose. Dysfunction of β-cells has profound metabolic consequences leading to hyperglycemia and diabetes mellitus. β-cells are destroyed via autoimmune reaction in type 1 diabetes (T1D). Type 2 diabetes (T2D), characterized by impaired β-cell functions and reduced insulin sensitivity, accounts for 90% of all diabetic patients. Islet transplantation is a promising treatment for T1D. Pluripotent stem cells provide an unlimited cell source to generate new β-cells for patients with T1D. Furthermore, derivation of induced pluripotent stem cells (iPSCs) from patients captures "disease-in-a-dish" for autologous cell replacement therapy, disease modeling, and drug screening for both types of diabetes. This review highlights essential steps in pancreas development, and potential stem cell applications in cell regeneration therapy for diabetes mellitus.     

Generation of genetically modified rats from embryonic stem cells

At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/enhancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rho-associated kinase, mitogen-activated protein kinase, TGF-β, and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germline transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases.     

Combined hydrogels that switch human pluripotent stem cells from self-renewal to differentiation

The ability of materials to define the architecture and microenvironment experienced by cells provides new opportunities to direct the fate of human pluripotent stem cells (HPSCs) [Robinton DA, Daley GQ (2012) Nature 481(7381):295–305]. However, the conditions required for self-renewal vs. differentiation of HPSCs are different, and a single system that efficiently achieves both outcomes is not available [Giobbe GG, et al. (2012) Biotechnol Bioeng 109(12):3119–3132]. We have addressed this dual need by developing a hydrogel-based material that uses ionic de-cross-linking to remove a self-renewal permissive hydrogel (alginate) and switch to a differentiation-permissive microenvironment (collagen). Adjusting the timing of this switch can preferentially steer the HPSC differentiation to mimic lineage commitment during gastrulation to ectoderm (early switch) or mesoderm/endoderm (late switch). As an exemplar differentiated cell type, we showed that directing early lineage specification using this single system can promote cardiogenesis with increased gene expression in high-density cell populations. This work will facilitate regenerative medicine by allowing in situ HPSC expansion to be coupled with early lineage specification within defined tissue geometries.Human pluripotent stem cells (HPSCs) comprise human embryonic stem cells (HESCs) and human induced pluripotent stem cells (1). The ability to couple expansion and differentiation of these cells underpins current efforts in regenerative medicine (2, 3). Initial efforts to direct the fate of HPSCs by recapitulating the developmental process of gastrulation [by using spontaneous differentiation of embryoid bodies (EBs)] have been refined to allow directed differentiation in two and three dimensions (3D) (4). This process includes coupling bioreactor expansion of HPSCs in 3D aggregates with differentiation to neural lineages (5). The differentiated cells from these processes can be harvested and then used to seed geometrically complex scaffolds. However, this two-stage process could be better controlled and streamlined by in situ HPSC expansion and differentiation within a single template. Furthermore, in situ tissue development more closely recapitulates embryogenesis (6) and could produce tissue with authentic cellular complexity and physiology (7).To date, natural (8, 9) and synthetic (10) materials have been developed to retain the self-renewal phenotype of HPSCs. We (11) and others (12) have shown that hydrogel systems can instruct cell behavior by providing cell-adhesive or nonadhesive microenvironments. Extracellular matrix (ECM) hydrogels, such as collagen, have fibrous microstructures (13) and are suitable for cell adhesion, growth, and migration (14). This characteristic is unlike hydrogels such as alginate, which are nonadhesive and nanoporous and prevent migration. Collagen (type I) is cross-linked by neutralizing acidity and leads to fibril formation, whereas alginate gels are formed or disaggregated by regulating divalent cation availability—usually Ca²⁺ in the form of CaCl₂ (15).Importantly, HPSC self-renewal and triggering of gastrulation-like differentiation requires different culture and microenvironmental conditions (2). Therefore, the development of hydrogel systems that allow the modification of the structural and adhesive microenvironment after the initial cross-linking would be ideal to control cell behavior (16–18). A previous study explored this concept and used alginate switching from cross-linked to un-cross-linked states to demonstrate nonadhesive-to-adhesive tailoring of the microenvironment in the presence of somatic cell lines. This switch affected attributes such as rate of solute transport, gel mechanics, cell adhesion, morphology, and migration (12). Here we describe the development of a system that can direct HPSC fate from self-renewal to differentiation by using alginate cross-linked/de-cross-linked state as a microenvironmental switch (Fig. 1A).Open in a separate windowFig. 1.(A) Alginate serves as structural modulator to prevent the adhesive and fibrous network created by collagen. Upon switching (chelation of Ca²⁺ ions), alginate is removed, and collagen fibers are generated, forming an adhesive microenvironment. (B) The 3D-printed gels retain geometry when switched by chelation (grid: 1 mm). (C) Quantitation of alginate and collagen before and after switching (n = 3). (D) Spectroscopy measuring collagen fiber character during cross-linking and switching of hydrogels. Alginate prevents complete collagen network formation until chelation and washout (n = 6). (E) Confocal images of collagen fibers with transmission imaging. Collagen fiber formation is inhibited until alginate is removed. (Bar: 20 µm.)     

Strategies to produce hepatocytes and hepatocyte-like cells from pluripotent stem cells

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a potent source for unlimited production of hepatocytes and hepatocyte-like cells that may replace primary human hepatocytes in a variety of fields including liver cell therapy, liver tissue engineering, manufacturing bioartificial liver, modeling inherited and chronic liver diseases, drug screening and toxicity testing. Human ESCs are able to spontaneously form embryoid bodies, which then spontaneously differentiate to various tissue-specific cell lineages containing a total of 10-30% albumin-producing hepatocytes and hepatocyte-like cells. Enrichment of embryoid bodies with the definitive endoderm, from which hepatocytes arise, yields increasing the final ratio of hepatocyte population up by 50-65%. Current strategies of the directed differentiation of human ESCs (and iPSCs) to hepatocytes that reproduce liver embryogenesis by sequential stimulation of culturing ESCs with tissue-specific growth factors result in achieving the differentiation rate up to 60-80%. In the future, directed differentiation of human ESCs and iPSCs to hepatocytes should be further optimized towards generating homogeneous cultures of hepatocytes in order to avoid expensive procedures of separation and isolation of hepatocytes and hepatocyte-like cells.     

鼠胚胎干细胞体外分化的单个心肌细胞获取方法

用鼠胚胎干细胞在体外分化成心肌细胞是研究心肌细胞进化和生理学实验的新途径 ,胚胎干细胞最初是从鼠胚胎在胚泡阶段或 8个细胞的胚胎阶段的内部没分化的细胞中得到。其可以定向诱导分化为几乎所有种类的细胞 ,此文介绍了由鼠胚胎干细胞分化成为心肌细胞及单个心肌细胞的获取方法。     

Insulin expressing cells from differentiated embryonic stem cells are not beta cells

Aim/hypothesis Embryonic stem (ES) cells have been proposed as a potential source of tissue for transplantation for the treatment of Type 1 diabetes. However, studies showing differentiation of beta cells from ES cells are controversial. The aim of this study was to characterise the insulin-expressing cells differentiated in vitro from ES cells and to assess their suitability for the treatment of diabetes.Methods ES cell-derived insulin-expressing cells were characterised by means of immunocytochemistry, RT-PCR and functional analyses. Activation of the Insulin I promoter during ES-cell differentiation was assessed in ES-cell lines transfected with a reporter gene. ES cell-derived cultures were transplanted into STZ-treated SCID-beige mice and blood glucose concentrations of diabetic mice were monitored for 3 weeks.Results Insulin-stained cells differentiated from ES cells were devoid of typical beta-cell granules, rarely showed immunoreactivity for C-peptide and were mostly apoptotic. The main producers of proinsulin/insulin in these cultures were neurons and neuronal precursors and a reporter gene under the control of the insulin I promoter was activated in cells with a neuronal phenotype. Insulin was released into the incubation medium but the secretion was not glucose-dependent. When the cultures were transplanted in diabetic mice they formed teratomas and did not reverse the hyperglycaemic state.Conclusions/Interpretation Our studies show that insulin-positive cells in vitro-differentiated from ES cells are not beta cells and suggest that alternative protocols, based on enrichment of ES cell-derived cultures with cells of the endodermal lineage, should be developed to generate true beta cells for the treatment of diabetes.Abbreviations ES Embryonic stem - LIF leukemia inhibitory factor - ITSF insulin-transferrin-selenite-fibronectin.Bleackley and Korbutt laboratories contributed equally to this paper     

Mesenchymal stem cells obtained after bone marrow transplantation or peripheral blood stem cell transplantation originate from host tissue

Mesenchymal stem cells (MSC) obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Therefore, we were interested in finding out if graft MSC can be detected in leukapheresis products and in bone marrow after BMT and peripheral blood stem cell transplantation (PBSCT). Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of nine leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for origin of MSC. Almost all MSC samples exhibited a complete host profile, whereas peripheral blood cells were of donor origin. We conclude that even if trace amounts of MSC are co-transplanted during PBSCT or BMT, they do not expand significantly in the host bone marrow.     

Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

AIM: To investigate the ability of a genetically altered embryonic stem (ES) cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 gene. METHODS: Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body (EB) culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone (DTZ), a zinc-chelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing cells. The outgrowths were incubated in DTZ solution (final concentration, 100 μg/mL) for 15 min before being examined microscopically. Gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ELISA. RESULTS: DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells (Nkx-ES cells), which was as much as 2 wk earlier, than those in the culture of parental ES cells (wt-ES). The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 (PDX1), proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular clusters. Insulin secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not demonstrated. CONCLUSION: Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.     

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

Aims/hypothesis We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors. Methods Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.Results The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.Conclusions/interpretation These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.Abbreviations Anti-Shh antibody against sonic hedgehog - D3 undifferentiated D3 stem cell line - EB embryoid bodies - ES embryonic stem - FBS fetal bovine serum - LIF leukaemia inhibitory factor - mES mouse embryonic stem - Ngn3 neurogenin 3 - P gelatine-coated plates - Pdx-1 pancreatic duodenum homeobox 1     

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM: To enhance the differentiation of insulin producing cell (IPC) ability from embryonic stem (ES) cells in vitro. METHODS: Four-day embryoid body (EB)-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator (Amaxa biosystems, Germany) in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR (SQ-PCR) detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixl1, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry (IHC), and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease (SCID) pretreated with streptozotocin (STZ) were used to eliminate plasma glucose restoration after pax4+ ES implantation. RESULTS: A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixl1, pdx1, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION: Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasmid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, thepotential of pax-4-nucleofected cells in medical treatment is promising.     

Cotransplantation of HLA-identical mesenchymal stem cells and hematopoietic stem cells in Chinese patients with hematologic diseases

Mesenchymal stem cells (MSCs) may be employed to support hematopoietic reconstitution and mitigate graft-vs.-host disease (GVHD) in transplantation of hematopoietic stem cells (HSCs). The aim of this study was to explore the feasibility and safety of cotransplantation culture-expanded MSCs and HSCs from the same human leukocyte antigen (HLA)-identical sibling donor in Chinese patients with hematologic diseases. Bone marrow mononuclear cells from healthy donors were cultured and expanded ex vivo. Immunophenotype, adipogenic and osteogenic differentiation potential, and karyotype of the harvested MSCs were detected on those who had been cotransplanted with HSCs and MSCs from the same donor. Hematopoietic reconstitutions, complications, and clinical outcomes were observed after cotransplantation in these patients. (1.77 ± 0.40) × 10⁶/kg (donor’s weight) MSCs were successfully expanded from 23.6 ± 5.96 ml of bone marrow samples. They had normal karyotypes with bi-lineages differentiation potential, and were CD73, CD90, and CD105 positive. Twelve patients underwent cotransplantation with no observable adverse response during and after the infusion of MSCs. Hematopoietic reconstitutions were rapid. Two patients developed grade II–IV acute GVHD, and two extensive chronic GVHD. Four patients suffered from cytomegalovirus infection but were cured eventually. Up to now, seven patients have been followed as long as 29–57 months and five patients died. It is concluded that MSCs can be expanded effectively by culture and it is safe and feasible to cotransplant patients with allogenic culture-expanded MSCs and HSCs.     

Therapeutic potential of embryonic stem cells

Nearly 20 years after murine embryonic stem cells (mESC) were isolated, the first report of the derivation of human embryonic stem cells (hESC) in 1998 spawned the field of hESC research [Evans MJ, Kaufman MH, Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292 (5819): 154-6; Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282 (5391): 1145-7.]. Although this field is only in its infancy, hESC represent a theoretically inexhaustible source of precursor cells that could be differentiated into any cell type to treat degenerative, malignant, or genetic diseases, or injury due to inflammation, infection, and trauma. This pluripotent, endlessly dividing cell has been hailed as a possible means for treating diabetes, Parkinson's disease, Alzheimer's, spinal cord injury, heart failure, and bone marrow failure. But the regenerative medicine applications of embryonic stem cells are only one facet of hESC therapeutic potential. Human ESC are an invaluable research tool to study development, both normal and abnormal, and can serve as a platform to develop and test new therapies. In addition to discussing the therapeutic potential of hESC, this chapter will cover limitations to using hESC for replacement cell therapy, strategies to overcome these limitations, and alternative methods of deriving hESC.     

Human embryonic stem cells and liver diseases: from basic research to future clinical application

Human embryonic stem cells (hESC) provide access to the earliest stages of human development and because of their high proliferation capability, pluripotency and low immunogenicity may serve as a potential source of specialized cells for regenerative medicine. hESC-derived hepatocyte-like cells exhibit characteristic hepatocyte morphology, express hepatocyte markers and are capable of executing a range of hepatocyte functions. However, there are many challenges and obstacles to be overcome before the use of hESC and hESC-derived hepatocyte-like cells in clinical practice can be realized. Here, we highlight some of the recent efforts in this area, in hope of providing insights toward this complex yet important area of therapeutical modality for treating patients with liver disease.     

Development and functional analysis of eosinophils from murine embryonic stem cells

We have established a culture system for the development of eosinophils from murine embryonic stem (ES) cells. After transferring ES cells from embryonic fibroblast cells onto macrophage colony-stimulating factor-deficient stromal cells, OP9, ES cells were cultured in the presence of interleukin (IL)-5 with either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF) for 20 d to obtain approximately 50% eosinophils. Electron microscopy confirmed the presence of crystallized major basic protein (MBP) in the granules of some of these cells. Neither IL-5, IL-3, GM-CSF nor eotaxin alone could induce eosinophils as efficiently as the conditions described above. Eotaxin induced eosinophil development in combination with either IL-3 or IL-5. Levels of GATA-1, Friend of GATA (FOG)-1, PU.1, CCAAT/enhancer binding protein (C/EBP)alpha, C/EBPbeta, IL-3 receptor alpha (IL-3Ralpha), GM-CSF receptor alpha (GM-CSFRalpha), and MBP mRNAs were increased in ES cells 10 d after transfer onto OP9 cells. In contrast, C/EBPepsilon, IL-5Ralpha, and eosinophil peroxidase mRNAs were induced in response to IL-3 and IL-5 after transfer onto OP9 cells. Eosinophils that developed in this system expressed Gr-1, F4/80, B220, CCR3, IL-3Ralpha, IL-5Ralpha, and DX5. Finally, eosinophils developed from ES cells produced reactive oxygen species in response to Leishmania as do peripheral blood eosinophils.     

Engineering bone tissue substitutes from human induced pluripotent stem cells

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.