Improved hematopoietic differentiation of human pluripotent stem cells via estrogen receptor signaling pathway

Background

Aside from its importance in reproduction, estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However, the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs).

Results

E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-α)-dependent pathway. During hematopoietic differentiation of hPSCs, ER-α is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-α positive cells. Interestingly, continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-α selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors, and subsequent erythropoiesis, whereas ER-β selective agonist did not. Furthermore, ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods, does E2 signaling potentiate hematopoietic development, suggesting universal function of E2 on hematopoiesis.

Conclusions

Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions.

     

Thalidomide induces apoptosis during early mesodermal differentiation of human induced pluripotent stem cells

Thalidomide was once administered to pregnant women as a mild sedative; however, it was subsequently shown to be strongly teratogenic. Recently, there has been renewed interest in thalidomide because of its curative effects against intractable diseases. However, the teratogenicity of thalidomide is manifested in various ways and is still not fully understood. In the present study, we evaluated the effects of thalidomide on early mesodermal differentiation by examining the differentiation of human induced pluripotent stem cells (hiPSCs). The most common symptom of thalidomide teratogenicity is limb abnormality, which led us to hypothesize that thalidomide prevents early mesodermal differentiation. Therefore, mesodermal differentiation of hiPSCs was induced over a 6-d period. To induce early mesoderm differentiation, 1 d after seeding, the cells were incubated with the small molecule compound CHIR99021 for 3 d. Thalidomide exposure was initiated at the same time as CHIR99021 treatment. After 5 d of thalidomide exposure, the hiPSCs began expressing a mesodermal marker; however, the number of viable cells decreased significantly as compared to that of control cells. We observed that the proportion of apoptotic and dead cells increased on day 2; however, the proportion of dead cells on day 5 had decreased, suggesting that the cells were damaged by thalidomide during early mesodermal differentiation (days 0–2). Our findings may help elucidate the mechanism underlying thalidomide teratogenicity and bring us closer to the safe use of this drug.     

Optimizing neuronal differentiation of human pluripotent NT2 stem cells in monolayer cultures

Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity testing at different stages of maturation. However, differentiation of NT2 cells is not straightforward. There are different protocols available in the literature reporting varying results with regard to differentiation efficiency, expression of neuronal markers and morphological characteristics of differentiated cells. Yet, the efficiency of available protocols has not been systematically compared. To address this question, we quantified the number and size of cell cluster formed during differentiation using published and modified protocols and analyzed the abundance of neuronal and non‐neuronal expression markers using immunocytochemistry. In the course of the experiments we observed that differentiation results strongly depend on the cell density at differentiation‐initiation as well as on the type of used cell culture plastic ware. Based on those observations and the results from our comparative analysis, we created our own optimized and robust protocol that reproducibly reveals differentiated cells with high yield. We conclude that our method may be superior to differentiation of NT2 cells for systematic in vitro‐based primary screening for developmental toxicants and neurotoxicants at different stages of maturation over previous protocols used. Our approach will also contribute to reduce animal testing in the context of the 3Rs.     

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures.     

Cardiac differentiation of human pluripotent stem cells

Due to the extremely limited proliferative capacity of adult cardiomyocytes, human embryonic (pluripotent) stem cell derived cardiomyocytes (hESC-CMs) are currently almost the only reliable source of human heart cells which are suited to large-scale production. These cells have the potential for wide-scale application in drug discovery, heart disease research and cell-based heart repair. Embryonic atrial-, ventricular- and nodal-like cardiomyocytes can be obtained from differentiated human embryonic stem cells (hESCs). In recent years, several highly efficient cardiac differentiation protocols have been developed. Significant progress has also been made on understanding cardiac subtype specification, which is the key to reducing the heterogeneity of hESC-CMs, a major obstacle to the utilization of these cells in medical research and future cell-based replacement therapies. Herein we review recent progress in cardiac differentiation of hESCs and cardiac subtype specification, and discuss potential applications in drug screening and cell-based heart regeneration.     

Development of serum-free culture systems for human embryonic stem cells

Human embryonic stem cells, because of their unique combination of long-term self-renewal properties and pluripotency, are providing new avenues of investigation of stem cell biology and human development and show promise in providing a new source of human cells for transplantation therapies and pharmaceutical testing. Current methods of propagating these cells using combinations of mouse fibroblast feeder cultures and bovine serum components are inexpensive and, in general, useful. However, the systematic investigation of the regulation of self-renewal and the production of safer sources of cells for transplantation depends on the elimination of animal products and the use of defined culture conditions. Both goals are served by the development of serum-free culture methods for human embryonic stem cells.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

Delayed differentiation in embryonic stem cells and mesodermal progenitors in the absence of CtBP2

Mammalian embryonic stem cells (ESCs) are characterized by an ability to self-renew and give rise to each of the three germ layers. ESCs are a pluripotential source of numerous primitive progenitors and committed lineages and can make stoichiometric decisions leading to either asymmetric or symmetric cell division. Several genes have been identified as essential for maintenance of self-renewal, but few non-lineage specific genes have been identified as essential for differentiation. We selected the chromatin factor Ctbp2 from microarray data for its enriched expression in stem cells, in comparison to committed progenitors. RNA interference (RNAi) was used to knockdown gene expression in mouse ESCs and the potential for transduced cells to self-renew and differentiate was assessed in ESC and mesodermal assays. Here, we demonstrate an important role for Ctbp2 in stem cell maintenance and regulation of differentiation using an in vitro system. The knockdown of Ctbp2 increases the prevalence of ESCs in culture, delays differentiation induced by LIF withdrawal, and introduces developmental changes in mesodermal differentiation. A model is presented for the importance of Ctbp2 in maintaining a balance in decisions to self-renewal and differentiate.     

Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT(1)R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT(1)R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT(1)R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.     

Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

Background aims

Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.

Developments in cell culture systems for human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications.     

Basal serum-free culture system which supports growth of fetal rat adrenal cells in primary monolayer cell culture

A serum-free culture system has been developed which allows primary cultures of fetal rat adrenal cells to divide in response to insulin 10 micrograms/l, yet retain their ability to respond to adrenocorticotropic hormone (ACTH) by increased corticosterone production. The essential components of this culture system are: (1) a high initial plating number of 125-130 x 10(3) cells/cm2; (2) a low oxygen concentration of 2.5% O2; (3) an initial plating period of 48 h in greater than or equal to 5% (v/v) fetal bovine serum. In contrast to reported requirements for the serum-free growth of adult adrenal cells, the fetal adrenal cells do not require the provision of any special matrix or a competence factor for serum-free growth or steroidogenic activity. This suggests that they are capable of rapidly generating their own matrix and competence factor in primary culture.     

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.     

Differentiation of serum-free embryoid bodies from human induced pluripotent stem cells into networks

Three-dimensional aggregation cultures allow for complex development of differentiated human induced pluripotent stem cells. However, this approach is not easily amenable to live-cell imaging and electrophysiological applications due to the thickness and the geometry of the tissue. Here, we present an improvement on the traditional aggregation method by combining the use of cell culture inserts with serum-free embryoid bodies (SFEBs). The use of this technique allows the structures to maintain their three-dimensional structure while thinning substantially. We demonstrate that this technique can be used for electrophysiological recodings as well as live-cell calcium imaging combined with electrical stimulation, akin to organotypic slice preparations. This provides an important experimental tool that can be used to bridge 3-D structures with traditional monolayer approaches used in stem cell applications.