Generation of human melanocytes from induced pluripotent stem cells

Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells

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Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.     

Differences in the microrheology of human embryonic stem cells and human induced pluripotent stem cells

Embryonic and adult fibroblasts can be returned to pluripotency by the expression of reprogramming genes. Multiple lines of evidence suggest that these human induced pluripotent stem (hiPS) cells and human embryonic stem (hES) cells are behaviorally, karyotypically, and morphologically similar. Here we sought to determine whether the physical properties of hiPS cells, including their micromechanical properties, are different from those of hES cells. To this end, we use the method of particle tracking microrheology to compare the viscoelastic properties of the cytoplasm of hES cells, hiPS cells, and the terminally differentiated parental human fibroblasts from which our hiPS cells are derived. Our results indicate that although the cytoplasm of parental fibroblasts is both viscous and elastic, the cytoplasm of hiPS cells does not exhibit any measurable elasticity and is purely viscous over a wide range of timescales. The viscous phenotype of hiPS cells is recapitulated in parental cells with disassembled actin filament network. The cytoplasm of hES cells is predominantly viscous but contains subcellular regions that are also elastic. This study supports the hypothesis that intracellular elasticity correlates with the degree of cellular differentiation and reveals significant differences in the mechanical properties of hiPS cells and hES cells. Because mechanical stimuli have been shown to mediate the precise fate of differentiating stem cells, our results support the concept that stem cell “softness” is a key feature of force-mediated differentiation of stem cells and suggest there may be subtle functional differences between force-mediated differentiation of hiPS cells and hES cells.     

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC-CM), which exhibit cardiac-like transmembrane action potentials, intracellular Ca(2+) transients and contractions. While major features of the excitation-contraction coupling of iPSC-CM have been well-described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31-day-old (post-plating) iPSC-CM generated from human hair follicle keratinocytes (HFKT-iPSC-CM) were analysed by electron microscopy, and compared with those of human embryonic stem-cell-derived cardiomyocytes (hESC-CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT-iPSC-CM and hESC-CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT-iPSC-CM. We also found in both cell types: (1) 'Ca(2+)-release units', which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.     

Generation of induced pluripotent stem cells from the prairie vole

The vast majority of animals mate more or less promiscuously. A few mammals, including humans, utilize more restrained mating strategies that entail a longer term affiliation with a single mating partner. Such pair bonding mating strategies have been resistant to genetic analysis because of a lack of suitable model organisms. Prairie voles are small mouse-like rodents that form enduring pair bonds in the wild as well as in the laboratory, and consequently they have been used widely to study social bonding behavior. The lack of targeted genetic approaches in this species however has restricted the study of the molecular and neural circuit basis of pair bonds. As a first step in rendering the prairie vole amenable to reverse genetics, we have generated induced pluripotent stem cell (IPSC) lines from prairie vole fibroblasts using retroviral transduction of reprogramming factors. These IPSC lines display the cellular and molecular hallmarks of IPSC cells from other organisms, including mice and humans. Moreover, the prairie vole IPSC lines have pluripotent differentiation potential since they can give rise to all three germ layers in tissue culture and in vivo. These IPSC lines can now be used to develop conditions that facilitate homologous recombination and eventually the generation of prairie voles bearing targeted genetic modifications to study the molecular and neural basis of pair bond formation.     

Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have gained enormous interest as potential sources for regenerative biomedical therapies and model systems for studying early development. Traditionally, mouse embryonic fibroblasts have been used as a supportive feeder layer for the sustained propagation of hPSCs. However, the use of nonhuman‐derived feeders presents concerns about the possibility of xenogenic contamination, labor intensiveness, and variability in experimental results in hPSC cultures. Toward addressing some of these concerns, we report the propagation of three different hPSCs on feeder‐free extracellular matrix (ECM)‐based substrates derived from human fibroblasts. hPSCs propagated in this setting were indistinguishable by multiple criteria, including colony morphology, expression of pluripotency protein markers, trilineage in vitro differentiation, and gene expression patterns, from hPSCs cultured directly on a fibroblast feeder layer. Further, hPSCs maintained a normal karyotype when analyzed after 15 passages in this setting. Development of this ECM‐based culture system is a significant advance in hPSC propagation methods as it could serve as a critical component in the development of humanized propagation systems for the production of stable hPSCs and its derivatives for research and therapeutic applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010