Contribution of retinoid X receptor signaling to the specification of skeletal muscle lineage

Pluripotent stem cells possess a tremendous potential for the treatment of many diseases because of their capacity to differentiate into a variety of cell lineages. However, they provide little promise for muscle-related diseases, mainly because of the lack of small molecule inducers to efficiently direct myogenic conversion. Retinoic acid, acting through the retinoic acid receptor (RAR) and retinoid X receptor (RXR), affects stem cell fate determination in a concentration-dependent manner, but it only has a modest efficacy on the commitment of ES cells into skeletal muscle lineage. The RXR is very important for embryonic development but is generally considered to act as a silent partner of RAR in a non-permissive mode. In this study, we have examined whether activation of the RXR by rexinoid or RXR-specific signaling play a role in the specification of stem cells into muscle lineage. Our findings demonstrate that mouse ES cells generate skeletal myocytes effectively upon treatment with rexinoid at the early stage of differentiation and that on a molecular level, rexinoid-enhanced myogenesis simulates the sequential events observed in vivo. Moreover, RXR-mediated myogenic conversion requires the function of β-catenin but not RAR. Our studies establish the feasibility of applying the RXR agonist in cell-based therapies to treat muscle-related diseases. The aptitude of mouse ES cells to generate skeletal myocytes following rexinoid induction also provides a model system to study the convergence of different signaling pathways in myogenesis.     

Efficient and Rapid Induction of Human iPSCs/ESCs into Nephrogenic Intermediate Mesoderm Using Small Molecule-Based Differentiation Methods

The first step in developing regenerative medicine approaches to treat renal diseases using pluripotent stem cells must be the generation of intermediate mesoderm (IM), an embryonic germ layer that gives rise to kidneys. In order to achieve this goal, establishing an efficient, stable and low-cost method for differentiating IM cells using small molecules is required. In this study, we identified two retinoids, AM580 and TTNPB, as potent IM inducers by high-throughput chemical screening, and established rapid (five days) and efficient (80% induction rate) IM differentiation from human iPSCs using only two small molecules: a Wnt pathway activator, CHIR99021, combined with either AM580 or TTNPB. The resulting human IM cells showed the ability to differentiate into multiple cell types that constitute adult kidneys, and to form renal tubule-like structures. These small molecule differentiation methods can bypass the mesendoderm step, directly inducing IM cells by activating Wnt, retinoic acid (RA), and bone morphogenetic protein (BMP) pathways. Such methods are powerful tools for studying kidney development and may potentially provide cell sources to generate renal lineage cells for regenerative therapy.     

Dorsomorphin, a selective small molecule inhibitor of BMP signaling, promotes cardiomyogenesis in embryonic stem cells

BACKGROUND: Pluripotent embryonic stem (ES) cells, which have the capacity to give rise to all tissue types in the body, show great promise as a versatile source of cells for regenerative therapy. However, the basic mechanisms of lineage specification of pluripotent stem cells are largely unknown, and generating sufficient quantities of desired cell types remains a formidable challenge. Small molecules, particularly those that modulate key developmental pathways like the bone morphogenetic protein (BMP) signaling cascade, hold promise as tools to study in vitro lineage specification and to direct differentiation of stem cells toward particular cell types. METHODOLOGY/ PRINCIPAL FINDINGS: We describe the use of dorsomorphin, a selective small molecule inhibitor of BMP signaling, to induce myocardial differentiation in mouse ES cells. Cardiac induction is very robust, increasing the yield of spontaneously beating cardiomyocytes by at least 20 fold. Dorsomorphin, unlike the endogenous BMP antagonist Noggin, robustly induces cardiomyogenesis when treatment is limited to the initial 24-hours of ES cell differentiation. Quantitative-PCR analyses of differentiating ES cells indicate that pharmacological inhibition of BMP signaling during the early critical stage promotes the development of the cardiomyocyte lineage, but reduces the differentiation of endothelial, smooth muscle, and hematopoietic cells. CONCLUSIONS/ SIGNIFICANCE: Administration of a selective small molecule BMP inhibitor during the initial stages of ES cell differentiation substantially promotes the differentiation of primitive pluripotent cells toward the cardiomyocytic lineage, apparently at the expense of other mesodermal lineages. Small molecule modulators of developmental pathways like dorsomorphin could become versatile pharmacological tools for stem cell research and regenerative medicine.     

Hypoxia inhibits differentiation of lineage-specific Rcho-1 trophoblast giant cells

Defects in placental development lead to pregnancies at risk for miscarriage and intrauterine growth retardation and are associated with preeclampsia, a leading cause of maternal death and premature birth. In preeclampsia, impaired placental formation has been associated with alterations in a specific trophoblast lineage, the invasive trophoblast cells. In this study, an RT-PCR Trophoblast Gene Expression Profile previously developed by our laboratory was utilized to examine the lineage-specific gene expression of the rat Rcho-1 trophoblast cell line. Our results demonstrated that Rcho-1 cells represent an isolated, trophoblast population committed to the giant cell lineage. RT-PCR analysis revealed that undifferentiated Rcho-1 cells expressed trophoblast stem cell marker, Id2, and trophoblast giant cell markers. On differentiation, Rcho-1 cells downregulated Id2 and upregulated Csh1, a marker of the trophoblast giant cell lineage. Neither undifferentiated nor differentiated Rcho-1 cells expressed spongiotrophoblast marker Tpbpa or labyrinthine markers Esx1 and Tec. Differentiating Rcho-1 cells in hypoxia did not alter the expression of lineage-specific markers; however, hypoxia did inhibit the downregulation of the trophoblast stem cell marker Id2. Differentiation in hypoxia also blocked the induction of CSH1 protein. In addition, hypoxia inhibited stress fiber formation and abolished the induction of palladin, a protein associated with stress fiber formation and focal adhesions. Thus, Rcho-1 cells can be maintained as a proliferative, lineage-specific cell line that is committed to the trophoblast giant cell lineage on differentiation in both normoxic and hypoxic conditions; however, hypoxia does inhibit aspects of trophoblast giant cell differentiation at the molecular, morphological, and functional levels.     

Calcineurin-NFAT signaling critically regulates early lineage specification in mouse embryonic stem cells and embryos

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.     

Bone regeneration via skeletal cell lineage plasticity: All hands mobilized for emergencies

An emerging concept is that quiescent mature skeletal cells provide an important cellular source for bone regeneration. It has long been considered that a small number of resident skeletal stem cells are solely responsible for the remarkable regenerative capacity of adult bones. However, recent in vivo lineage‐tracing studies suggest that all stages of skeletal lineage cells, including dormant pre‐adipocyte‐like stromal cells in the marrow, osteoblast precursor cells on the bone surface and other stem and progenitor cells, are concomitantly recruited to the injury site and collectively participate in regeneration of the damaged skeletal structure. Lineage plasticity appears to play an important role in this process, by which mature skeletal cells can transform their identities into skeletal stem cell‐like cells in response to injury. These highly malleable, long‐living mature skeletal cells, readily available throughout postnatal life, might represent an ideal cellular resource that can be exploited for regenerative medicine.     

Cell lineage determination in the mouse

During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.     

Cell-surface proteomics identifies lineage-specific markers of embryo-derived stem cells

The advent of reprogramming and its impact on stem cell biology has renewed interest in lineage restriction in mammalian embryos, the source of embryonic (ES), epiblast (EpiSC), trophoblast (TS), and extraembryonic endoderm (XEN) stem cell lineages. Isolation of specific cell types during stem cell differentiation and reprogramming, and also directly from embryos, is a major technical challenge because few cell-surface proteins are known that can distinguish each cell type. We provide a large-scale proteomic resource of cell-surface proteins for the four embryo-derived stem cell lines. We validated 27 antibodies against lineage-specific cell-surface markers, which enabled investigation of specific cell populations during ES-EpiSC reprogramming and ES-to-XEN differentiation. Identified markers also allowed prospective isolation and characterization of viable lineage progenitors from blastocysts by flow cytometry. These results provide a comprehensive stem cell proteomic resource and enable new approaches to interrogate the mechanisms that regulate cell fate specification.     

Guiding embryonic stem cells towards differentiation: lessons from molecular embryology

Embryonic stem cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types, including germ cells. These qualities have made mouse embryonic stem cells a valuable resource for genetic manipulation of the mouse genome. In addition, they present a powerful system for the in vitro dissection of mammalian embryonic development. The recent isolation of human embryonic stem cells has raised a lot of interest for the potential of transposing our knowledge of lineage-specific differentiation of embryonic stem cells to cell-based therapy of human disease. Recent reports have provided insights into the specific differentiation of embryonic stem cells to different cell types of the embryo. However, progress in this direction seems to depend on the knowledge of the mechanisms controlling lineage decisions during embryogenesis.     

Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment

DNA methylation has been implicated as an epigenetic component of mechanisms that stabilize cell-fate decisions. Here, we have characterized the methylomes of human female hematopoietic stem/progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with lineage-specific genes were often methylated in the opposing lineage. In HSPCs, these sites tended to show intermediate, complex patterns that resolve to uniformity upon differentiation, by increased or decreased methylation. Promoter HMRs shared across diverse cell types typically display a constitutive core that expands and contracts in a lineage-specific manner to fine-tune the expression of associated genes. Many newly identified intergenic HMRs, both constitutive and lineage specific, were enriched for factor binding sites with an implied role in genome organization and regulation of gene expression, respectively. Overall, our studies represent an important reference data set and provide insights into directional changes in DNA methylation as cells adopt terminal fates.     

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.     

Myogenic and neurogenic differentiation of human tooth germ stem cells (hTGSCs) are regulated by pluronic block copolymers

Stem cells with high proliferation, self-renewal and differentiation capacities are promising for tissue engineering approaches. Among stem cells, human tooth germ stem cells (hTGSCs) having mesenchymal stem cell characteristics are highly proliferative and able to differentiate into several cell lineages. Researchers have recently focused on transplanting stem cells with bioconductive and/or bioinductive materials that can provide cell commitment to the desired cell lineages. In the present study, effects of pluronic block copolymers (F68, F127 and P85) on in vitro myo- and neurogenic differentiation of human tooth germ stem cells (hTGSCs) were investigated. As P85 was found to exert considerable toxicity to hTGSCs even at low concentrations, it was not evaluated for further differentiation experiments. Immunocytochemical analysis, gene and protein expression studies revealed that while F68 treatment increased lineage-specific gene expression in both myo- and neuro-genically differentiated cells, F127 did not result in any remarkable difference compared to cells treated with differentiation medium. Subsequent studies are required to explore the exact mechanisms of how F68 increases the myogenic and neurogenic differentiation of hTGSCs. The present work indicates that pluronic F68 might be used in functional skeletal and neural tissue engineering applications.     

小分子化合物诱导干细胞向视网膜感光细胞分化的研究进展

干细胞是一类具有多向分化潜能的细胞群,如胚胎干细胞(embryonic stem cell,ESC)、诱导多潜能干细胞(induced pluripotent stem cell,i PSC)等,可在特定的条件下向包括视网膜感光细胞在内的多种细胞分化。小分子化合物是一类由组织细胞合成、分泌的小分子多肽类因子,特定的小分子化合物可作用于干细胞诱导其向视网膜感光细胞分化。目前,对干细胞体外培养,通过使用不同的诱导培养方案,探索干细胞向视网膜感光细胞分化的研究成为热点。早期,研究者们主要在共培养条件下采用小分子化合物诱导ESC向视网膜感光细胞分化,随着研究的进展,逐渐开始探索在无共培养条件下小分子化合物诱导ESC向视网膜感光细胞的分化以及小分子化合物诱导i PSC向视网膜感光细胞的分化。本文主要就小分子化合物促进ESC和i PSC向视网膜感光细胞分化的研究进展进行综述。