Induced pluripotent stem cells for modelling human diseases

Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.     

Development of isolation and cultivation method for outer root sheath cells from human hair follicle and construction of bioartificial skin

Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites. Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS cells is 2.1×10³ cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified method was able to obtain about 6.9×10³ cells/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution. Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0×10⁷ cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total of 6.3×10⁷ cells over 17 days when starting with 7.5×10⁴ cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing the bioartificial skin epitheliumin vitro     

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.     

新物种诱导多能干细胞的研究进展

胚胎干细胞（embryonic stem cells,ESC）在人类遗传病学研究、疾病模型建立、器官再生以及动物物种改良和定向变异等方面的地位是其他类型的细胞不可取代的。但是,由于实验技术和体外培养条件的限制,除了小鼠、恒河猴和人之外,大鼠、猪、牛、羊等其他哺乳动物的ES细胞系被证明很难获得。先后有多个研究小组报道了他们利用新兴的诱导多能干细胞（induced pluripotent stem cells,iPS细胞）技术成功建立大鼠和猪的iPS细胞系的研究成果。迄今为止,这两个物种是在未成功建立ES细胞系之前利用iPS技术建立多能干细胞系的成功范例。这些研究对于那些还未建立ES细胞的物种建立多能干细胞系提供了一种新的方案,也将给这些物种的胚胎干细胞的建立、基因修饰动物的产生以及人类医疗事业的促进和发展带来新的希望。     

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs)

Introduction

(1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory.

Sources of data

We reviewed the literature related to reprogramming, pluripotency and fetal stem cells.

Areas of agreement

(1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both.

Areas of controversy

(1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The “level” of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined.

Growing points

Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.     

T lineage differentiation from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have potential to differentiate into T lymphocytes, however, the actual ability of iPS cells to develop into T lineages is not clear. In this study, we co-cultured iPS cells on OP9 cells expressing the Notch ligand Delta-like 1 (DL1), the iPS cells differentiated into T lymphocytes. In addition, in vitro stimulation of iPS cell-derived T lymphocytes resulted in secretion of IL-2 and IFN-γ. Moreover, adoptive transfer of iPS cell-derived T lymphocytes into Rag-deficient mice reconstituted their T cell pools. These results indicate that iPS cells are able to follow the normal program of T cell differentiation.     

Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation

Most mammals are coated with pigmented hair. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. The key to understanding the mechanism of cyclic melanin production is the melanocyte stem cell (MelSC) population, previously known as 'amelanotic melanocytes'. The MelSCs directly adhere to hair follicle stem cells, the niche cells for MelSCs and reside in the hair follicle bulge-subbulge area, the lower permanent portion of the hair follicle, to serve as a melanocyte reservoir for skin and hair pigmentation. MelSCs form a stem cell system within individual hair follicles and provide a 'hair pigmentary unit' for each cycle of hair pigmentation. This review focuses on the identification of MelSCs and their characteristics and explains the importance of the MelSC population in the mechanisms of hair pigmentation, hair greying, and skin repigmentation.     

Specific lectin biomarkers for isolation of human pluripotent stem cells identified through array-based glycomic analysis

Rapid and dependable methods for isolating human pluripotent stem cell (hPSC) populations are urgently needed for quality control in basic research and in cell-based therapy applications. Using lectin arrays, we analyzed glycoproteins extracted from 26 hPSC samples and 22 differentiated cell samples, and identified a small group of lectins with distinctive binding signatures that were sufficient to distinguish hPSCs from a variety of non-pluripotent cell types. These specific biomarkers were shared by all the 12 human embryonic stem cell and the 14 human induced pluripotent stem cell samples examined, regardless of the laboratory of origin, the culture conditions, the somatic cell type reprogrammed, or the reprogramming method used. We demonstrated a practical application of specific lectin binding by detecting hPSCs within a differentiated cell population with lectin-mediated staining followed by fluorescence microscopy and flow cytometry, and by enriching and purging viable hPSCs from mixed cell populations using lectin-mediated cell separation. Global gene expression analysis showed pluripotency-associated differential expression of specific fucosyltransferases and sialyltransferases, which may underlie these differences in protein glycosylation and lectin binding. Taken together, our results show that protein glycosylation differs considerably between pluripotent and non-pluripotent cells, and demonstrate that lectins may be used as biomarkers to monitor pluripotency in stem cell populations and for removal of viable hPSCs from mixed cell populations.     

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.     

Generation of functional gut-like organ from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have the pluripotency to differentiate into broad spectrum derivatives of all three embryonic germ layers. However, the in vitro organ differentiation potential of iPS cells to organize a complex and functional “organ” has not yet been demonstrated. Here, we demonstrate that mouse iPS cells have the ability to organize a gut-like organ with motor function in vitro by a hanging drop culture system. This “induced gut (iGut)” exhibited spontaneous contraction and highly coordinated peristalsis accompanied by a transportation of contents. Ultrastructural analysis identified that the iGut had large lumens surrounded by three distinct layers (epithelium, connective tissue and musculature). Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICCs, enteric pacemaker cells), was observed in the wall of the lumen and formed a distinct and dense network. The neurofilament immunoreactivity was identified to form large ganglion-like structures and dense neuronal networks. The iGut was composed of all the enteric components of three germ layers: epithelial cells (endoderm), smooth muscle cells (mesoderm), ICCs (mesoderm), and enteric neurons (ectoderm). This is the first report to demonstrate the in vitro differentiation potential of iPS cells into particular types of functional “organs.” This work not only contributes to understanding the mechanisms of incurable gut disease through disease-specific iPS cells, but also facilitates the clinical application of patient-specific iPS cells for novel therapeutic strategies such as patient-specific “organ” regenerative medicine in the future.     

A simplified method to generate serotonergic neurons from mouse embryonic stem and induced pluripotent stem cells

We have developed a new simple method to induce serotonergic neurons from embryonic stem (ES) and induced pluripotent stem cells. When ES or induced pluripotent stem cells were cultured on a thick gel layer of Matrigel, most colonies extended TuJ1-positive neurites. We found that noggin, a known antagonist of bone morphogenic protein, induces ES cells to express genes involved in serotonergic differentiation, such as Nkx2.2, Pet-1, Sonic hedgehog, tryptophan hydroxylase 2, and serotonin transporter, as well as increases high potassium-induced release of serotonin. To concentrate serotonergic neurons, ES cells carrying Pet-1-enhancer-driven enhanced green fluorescent protein were differentiated and sorted into about 80% pure cultures of serotonergic neurons. Whole cell voltage-clamp recordings showed a voltage-dependent current in dissociated neurons. This simplified method provides an alternative option for serotonergic differentiation of pluripotent stem cells and will likely contribute a deeper understanding regarding the nature of serotonergic neurons and open new therapeutic perspectives for the treatment of psychiatric disorders.     

Organotypic culture of outer root sheath cells from human hair follicles using a new culture device

Summary A new culture vessel for the growth of cells on biological substrata and under organotypic conditions is described. This device, named Combi-ring-dish (CRD), is composed of four concentric rings designed to take up one or several substrata on which cells can be grown either immersed in culture medium or exposed to air and fed from underneath. Using the CRD, outer root sheath cells, isolated from plucked human hair follicles and plated on growth-arrested 3T3 feeder layers, were grown on native collagen lattices populated with living human fibroblasts. After reaching confluence, the immersed cultures were recombined (in vitro) with pieces of freeze-killed dermis and grown further, exposed to air. Thus by mimicking epidermal growth conditions, differentiation was dramatically improved, compared to control cultures on plastic substratum. Virtually all morphologic features of interfollicular epidermis developed. This seems a suitable model to investigate the differentiation potential of human hair follicle cells.     

Clonal expansion of human pluripotent stem cells on gelatin-coated surface

The research of human pluripotent stem cells is important for providing the molecular basis for their future application to regenerative medicine. To date, they are usually cultured on feeder cells and passaged by partial dissociation with either enzymatic or mechanical methods, which are problematic for the research using them in the convenience and reproducibility. Here we established a new culture system that allows handling as easily as culturing feeder-free mouse ES cells. This newly developed culture system is based on the combinatorial use of ROCK inhibitor and soluble fibronectin, which enables us to expand human pluripotent stem cells from single cell dissociation on gelatin-coated surface without any feeder cells. In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers. These cells also have abilities to differentiate into three germ layers in vivo and in vitro. Furthermore, no chromosomal abnormalities are found even after sequential passage. Therefore this system will dramatically simplify genetic engineering of these human pluripotent stem cells or defining process of their signal pathway.     

Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.