External stimulus-responsive biomaterials designed for the culture and differentiation of ES,iPS, and adult stem cells

The physical and chemical characteristics of biomaterial surface and hydrogels can be altered by external stimuli, such as light irradiation, temperature changes, pH shifts, shear stress forces, electrical forces, and the addition of small chemical molecules. Such external stimulus-responsive biomaterials represent promising candidates that have been developed for the culture and differentiation of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and adult stem cells. Biomaterials that are designed to respond in a reversible manner to specific external signals can be formed on micropatterned or non-micropatterned surface, in hydrogels, or on microcarriers. Stem cells and the cells differentiated from them into specific tissue lineages can be cultured and/or differentiated on dishes with immobilized external stimulus-responsive polymers. Cells can be detached from these dishes without using an enzymatic digestion method or a mechanical method when the appropriate external stimulus is generated on the surface. This review discusses the polymers and polymeric designs employed to produce surface and hydrogels for stem cell culture, differentiation, and/or cell detachment using various external stimuli.     

Polymeric design of cell culture materials that guide the differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the potential to differentiate into many cell types that originate from the three germ layers, such as dopamine-secreting cells and insulin-secreting cells for the treatment of Alzheimer's disease and diabetes, respectively. However, it is challenging to guide hPSC differentiation into desired cell lineages due to their varying differentiation ability. A reasonable strategy is to mimic the stem cell microenvironment for the differentiation of hPSCs into specific cell lineages using optimal polymeric biomaterials for hPSC culture. This review summarizes various methods for differentiating hPSCs cultured on polymeric biomaterials and discusses the optimal methods and cell culture polymeric biomaterials for hPSC differentiation into specific cell lineages. The recent trend in protocols avoids embryoid body (EB, aggregated cells) formation because EBs contain different types of cells. The combination of appropriate differentiation protocols and cell culture polymeric biomaterials for the differentiation of hPSCs into specific cell lineages will produce a large quantity of highly pure GMP-grade differentiated cells for use in translational medicine.     

DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.     

Generation of Bladder Urothelium from Human Pluripotent Stem Cells under Chemically Defined Serum- and Feeder-Free System

Human stem cells are promising sources for bladder regeneration. Among several possible sources, pluripotent stem cells are the most fascinating because they can differentiate into any cell type, and proliferate limitlessly in vitro. Here, we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE), and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes, but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs, they notably expressed urothelium-specific genes such as UPIb, UPII, UPIIIa, P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII, CK8/18 and P63 as well as tight junction molecules, E-CADHERIN and ZO-1. Additionally, hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay, indicating BUCs possessed the functional units of barrier on their surfaces. However, BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary, we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro.     

Apoptosis in Porcine Pluripotent Cells: From ICM to iPSCs

Pigs have great potential to provide preclinical models for human disease in translational research because of their similarities with humans. In this regard, porcine pluripotent cells, which are able to differentiate into cells of all three primary germ layers, might be a suitable animal model for further development of regenerative medicine. Here, we describe the current state of knowledge on apoptosis in pluripotent cells including inner cell mass (ICM), epiblast, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Information is focused on the apoptotic phenomenon in pluripotency, maintenance, and differentiation of pluripotent stem cells and reprogramming of somatic cells in pigs. Additionally, this review examines the multiple roles of apoptosis and summarizes recent progress in porcine pluripotent cells.     

A Non-Toxic Concentration of Telomerase Inhibitor BIBR1532 Fails to Reduce TERT Expression in a Feeder-Free Induced Pluripotent Stem Cell Model of Human Motor Neurogenesis

Several studies have shown that human induced pluripotent stem cell (iPSC)-derivatives are essentially fetal in terms of their maturational status. Inducing ageing in iPSC-motor neuron (MN) models of amyotrophic lateral sclerosis (ALS) has the potential to capture pathology with higher fidelity and consequently improve translational success. We show here that the telomerase inhibitor BIBR1532, hypothesised to recapitulate the telomere attrition hallmark of ageing in iPSC-MNs, was in fact cytotoxic to feeder-free iPSCs when used at doses previously shown to be effective in iPSCs grown on a layer of mouse embryonic fibroblasts. Toxicity in feeder-free cultures was not rescued by co-treatment with Rho Kinase (ROCK) inhibitor (Y-27632). Moreover, the highest concentration of BIBR1532 compatible with continued iPSC culture proved insufficient to induce detectable telomerase inhibition. Our data suggest that direct toxicity by BIBR1532 is the most likely cause of iPSC death observed, and that culture methods may influence enhanced toxicity. Therefore, recapitulation of ageing hallmarks in iPSC-MNs, which might reveal novel and relevant human disease targets in ALS, is not achievable in feeder-free culture through the use of this small molecule telomerase inhibitor.     

Maintenance of hemiround colonies and undifferentiated state of mouse induced pluripotent stem cells on carbon nanotube-coated dishes

Induced pluripotent stem (iPS) cells have attracted worldwide interest. However, there have been only a few studies investigating effective culture substrates for feeder-free culturing for the maintenance of iPS cells. In this study, we cultured mouse iPS cells under feeder-free conditions on carbon nanotube (CNT)-coated dishes and then evaluated the colony morphology and differentiation state of the cells on the dishes. After 5 d of cultivation in a medium containing 15% fetal bovine serum (FBS) and leukemia inhibitory factor (LIF), the colonies on thick films of multi-walled CNTs (MWCNTs) were observed to be hemiround; further, the cells expressed early undifferentiation markers. On the other hand, the colonies on a cell culture polystyrene dish and a collagen-coated polystyrene dish showed indistinct outline and spread well, and most spreading cells only weakly expressed early undifferentiation markers. These results indicate that the thick films of MWCNTs could maintain hemiround colonies and undifferentiated state of mouse iPS cells cultured under feeder-free conditions.     

Photoactivable Elimination of Tumorigenic Human Induced Pluripotent Stem Cells by Using a Lectin–Doxorubicin Prodrug Conjugate

Human pluripotent stem cells (hPSCs) are attractive resources for regenerative medicine, but medical applications are hindered by their tumorigenic potential. Previously, a hPSC-specific lectin probe, rBC2LCN, was identified through comprehensive glycome analysis by using high-density lectin microarrays. Herein, a lectin–doxorubicin (DOX) prodrug conjugate, with controllable photolysis activation for the elimination of tumorigenic human induced pluripotent stem cells, has been developed. rBC2LCN was fused with a biotin-binding protein, tamavidin (BC2Tama), and the fusion protein was expressed in Escherichia coli and purified by means of affinity chromatography. BC2Tama was then conjugated with doxorubicin-photocleavable biotin (DOXPCB). The BC2Tama–DOXPCB conjugates were observed to bind to hPSCs followed by internalization. Upon exposure to ultraviolet light, DOX was released inside the cells, which allowed specific killing of the hPSCs. Thus, BC2Tama–DOXPCB should be useful for the targeted elimination of hPSCs contained in hPSC-derived cell therapy products. This is the first report of the generation of lectin–prodrug conjugates. BC2Tama should be applicable for the targeted delivery of various types of biotinylated compounds into hPSCs.     

Hydrogels as smart biomaterials

Hydrogels were the first biomaterials rationally designed for human use. Beginning with the pioneering work of Wichterle and Lím on three‐dimensional polymers that swell in water, we review the design, synthesis, properties, and applications of hydrogels. The field of hydrogels has moved forward at a dramatic pace. The development of suitable synthetic methods encompassing traditional chemistry to molecular biology has been used in the design of hydrogels mimicking basic processes of living systems. Stimuli‐sensitive hydrogels, hydrogels with controlled degradability, genetically engineered poly(amino acid) polymers reversibly self‐assembling in precisely defined three‐dimensional structures, and hybrid polymers composed of two distinct classes of molecules are just some examples of these exciting novel biomaterials. The biocompatibility of hydrogels and their applications from implants to nanomaterials are also reviewed. Copyright © 2007 Society of Chemical Industry     

Epigenetic Regulation of Cardiomyocyte Differentiation from Embryonic and Induced Pluripotent Stem Cells

With the intent to achieve the best modalities for myocardial cell therapy, different cell types are being evaluated as potent sources for differentiation into cardiomyocytes. Embryonic stem cells and induced pluripotent stem cells have great potential for future progress in the treatment of myocardial diseases. We reviewed aspects of epigenetic mechanisms that play a role in the differentiation of these cells into cardiomyocytes. Cardiomyocytes proliferate during fetal life, and after birth, they undergo permanent terminal differentiation. Upregulation of cardiac-specific genes in adults induces hypertrophy due to terminal differentiation. The repression or expression of these genes is controlled by chromatin structural and epigenetic changes. However, few studies have reviewed and analyzed the epigenetic aspects of the differentiation of embryonic stem cells and induced pluripotent stem cells into cardiac lineage cells. In this review, we focus on the current knowledge of epigenetic regulation of cardiomyocyte proliferation and differentiation from embryonic and induced pluripotent stem cells through histone modification and microRNAs, the maintenance of pluripotency, and its alteration during cardiac lineage differentiation.     

诱导性多能干细胞免疫原性的研究进展

2006年,研究人员将一系列转录因子导入小鼠成纤维细胞中,诱导出了一种类似于胚胎干细胞状态的细胞,称为"诱导性多能干细胞"(induced pluripotent stem cell,简称iPS细胞)。iPS细胞在具有高度自我更新和分化能力的同时,进一步避免了传统胚胎干细胞的伦理学问题,特别是该技术使干细胞自体化移植更易实现,在很大程度上推进了干细胞技术的临床应用。目前,在iPS细胞的诱导方式和诱导效率方面,已取得了较大进展,但在iPS细胞的诱导过程以及再分化过程中,细胞针对于自体的免疫原性是否发生改变,目前尚不明确。本文就近年来iPS细胞免疫原性的研究进展作一简要综述。     

Generation of an ICF Syndrome Model by Efficient Genome Editing of Human Induced Pluripotent Stem Cells Using the CRISPR System

Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.     

Characteristics and Therapeutic Potential of Human Amnion-Derived Stem Cells

Stem cells including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs) are able to repair/replace damaged or degenerative tissues and improve functional recovery in experimental model and clinical trials. However, there are still many limitations and unresolved problems regarding stem cell therapy in terms of ethical barriers, immune rejection, tumorigenicity, and cell sources. By reviewing recent literatures and our related works, human amnion-derived stem cells (hADSCs) including human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial stem cells (hAESCs) have shown considerable advantages over other stem cells. In this review, we first described the biological characteristics and advantages of hADSCs, especially for their high pluripotency and immunomodulatory effects. Then, we summarized the therapeutic applications and recent progresses of hADSCs in treating various diseases for preclinical research and clinical trials. In addition, the possible mechanisms and the challenges of hADSCs applications have been also discussed. Finally, we highlighted the properties of hADSCs as a promising source of stem cells for cell therapy and regenerative medicine and pointed out the perspectives for the directions of hADSCs applications clinically.     

Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.     

Three‐dimensional hydrogel encapsulated embryonic stem and carcinoma cells as culture platforms for cytotoxicity studies

The utility of alginate hydrogels for three‐dimensional (3‐D) culture of mouse embryonic stem cells (mESCs) and future development of 3‐D stem cell culture‐based in vitro screens of toxicity is described. Using alginate hydrogels of various stiffness, we first evaluated the impact of substrate modulus on mESC viability, proliferation, as well as expression of pluripotency and germ‐layer markers and observed that low concentration alginate hydrogels (0.5% and 1% alginate) were most suitable for long‐term culture of mESCs. These results were not unique to mESCs; long‐term viability and proliferation of mouse embryonic carcinoma cells (mECCs) was also best supported by similar conditions. Finally, we determined cytotoxic responses of alginate encapsulated cells to commercially available chemicals and interestingly observed similar responses for mESCs and mECCs, thereby suggesting that mECCs can predict stem cell responses to chemicals. These studies will facilitate future design of optimal stem cell‐based platforms of organ‐specific and developmental toxicity. © 2015 American Institute of Chemical Engineers AIChE J, 61: 3180–3184, 2015     

Engineering neural stem cell fates with hydrogel design for central nervous system regeneration

Injuries and disease to the central nervous system (CNS) are accompanied by severe consequences, as the adult CNS has very limited capacity to replace the lost neural cells. Different sources of neural stem cells for CNS tissue regeneration exist, including embryonic stem cells (ESCs), fetal stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs), and so on. However, before stem cell therapy can be a viable option for treatments, many issues still need to be resolved, including low viability, lack of control of stem cell fate, and low cell engraftment after transplantation. Though controlling these parameters is extremely challenging, engineering structures that create permissive niches for the transplanted cells, such as the use of biocompatible hydrogels, is a promising approach. This review will focus on highlighting existing hydrogel systems currently being investigated for CNS tissue regeneration, as well as discuss design criteria for hydrogels and methods for manipulating stem cells within hydrogels systems. Finally, the use of these hydrogel systems as carriers for stem cell transplantation in CNS injury and disease models will be discussed.     

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs) generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF) cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1) and Rex-1 (ZFP-42, zinc finger protein 42). Using embryonic stem cells (ESCs) conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs) using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF) cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.     

Derivation of Mouse Parthenogenetic Advanced Stem Cells

Parthenogenetic embryos have been widely studied as an effective tool related to paternal and maternal imprinting genes and reproductive problems for a long time. In this study, we established a parthenogenetic epiblast-like stem cell line through culturing parthenogenetic diploid blastocysts in a chemically defined medium containing activin A and bFGF named paAFSCs. The paAFSCs expressed pluripotent marker genes and germ-layer-related genes, as well as being alkaline-phosphatase-positive, which is similar to epiblast stem cells (EpiSCs). We previously showed that advanced embryonic stem cells (ASCs) represent hypermethylated naive pluripotent embryonic stem cells (ESCs). Here, we converted paAFSCs to ASCs by replacing bFGF with bone morphogenetic protein 4 (BMP4), CHIR99021, and leukemia inhibitory factor (LIF) in a culture medium, and we obtained parthenogenetic advanced stem cells (paASCs). The paASCs showed similar morphology with ESCs and also displayed a stronger developmental potential than paAFSCs in vivo by producing chimaeras. Our study demonstrates that maternal genes could support parthenogenetic EpiSCs derived from blastocysts and also have the potential to convert primed state paAFSCs to naive state paASCs.