Integrated live imaging and molecular profiling of embryoid bodies reveals a synchronized progression of early differentiation.

Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live two-photon imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a \u27developmental clock\u27 using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model

Gingival Fibroblasts as a Promising Source of Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort.We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs) via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells) or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells) without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES) cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity.These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications. In addition, high-quality iPS cells were generated from mouse GFs without Myc transduction or a specific system for reprogrammed cell selection

An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20–100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo

Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming

SummaryReprogramming of somatic cells achieved by combination of the four transcription factors Oct4, Sox2, Klf4, and c-Myc has very low efficiency. To increase the reprogramming efficiency and better understand the process, we sought to identify factors that mediate reprogramming with higher efficiency. We established an assay to screen nuclear fractions from extracts of pluripotent mouse cells based on Oct4 reactivation. Using proteomics, we identified components of the ATP-dependent BAF chromatin-remodeling complex, which significantly increases reprogramming efficiency when used together with the four factors. The reprogrammed cells could transmit to the germline and exhibited pluripotency. Reprogramming remained highly efficient when c-Myc was not present but BAF components were overexpressed. BAF complex components mediate this effect by facilitating enhanced Oct4 binding to target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neural cells

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects

Capture of Neuroepithelial-Like Stem Cells from Pluripotent Stem Cells Provides a Versatile System for In Vitro Production of Human Neurons

Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) provide new prospects for studying human neurodevelopment and modeling neurological disease. In particular, iPSC-derived neural cells permit a direct comparison of disease-relevant molecular pathways in neurons and glia derived from patients and healthy individuals. A prerequisite for such comparative studies are robust protocols that efficiently yield standardized populations of neural cell types. Here we show that long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) derived from 3 hESC and 6 iPSC lines in two independent laboratories exhibit consistent characteristics including i) continuous expandability in the presence of FGF2 and EGF; ii) stable neuronal and glial differentiation competence; iii) characteristic transcription factor profile; iv) hindbrain specification amenable to regional patterning; v) capacity to generate functionally mature human neurons. We further show that lt-NES cells are developmentally distinct from fetal tissue-derived radial glia-like stem cells. We propose that lt-NES cells provide an interesting tool for studying human neurodevelopment and may serve as a standard system to facilitate comparative analyses of hESC and hiPSC-derived neural cells from control and diseased genetic backgrounds

Concise Review: Stem/progenitor cell proteoglycans decorated with 7-D-4, 4-C-3, and 3-B-3(-) chondroitin sulfate motifs are morphogenetic markers of tissue development

This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4‐C‐3, 7‐D‐4, and 3‐B‐3(‐), which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulfation motifs 7‐D‐4, 4‐C‐3, and 3‐B‐3 (‐) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. Stem Cells 201

Conversion of human fibroblasts to endothelial-like cells via dedifferentiation to mesodermal progenitors in 3D condensates

We have previously shown that human mesenchymal stromal cells dedifferentiate to early mesoderm when cultured in defined three-dimensional (3D) in vitro conditions, to mimic mesenchymal condensation, through a controlled autophagy response. Here it was determined if human dermal fibroblasts (HDFs), a terminally differentiated cell, could similarly undergo dedifferentiation. By varying initiating cell number and culture duration, we identified optimised conditions for HDF dedifferentiation in 3D spheroids. This was revealed by QPCR which identified low level expression of pluripotency factors, Oct4, Sox2 and Nanog, and a 6 to 1200-fold increase in mesodermal markers, Brachyury, Goosecoid and CXCR4, compared to adherent 2D HDFs, whilst expression of the CXCR4 ligand and stromal marker CXCL12 decreased. CXCR4 protein expression was confirmed by western blot analysis and immunostaining, which showed positive distribution across the spheroid. These changes occurred with a concomitant increase in autophagic features (increased TFEB mRNA expression, elevated LAMP-1 protein and the accumulation of autophagosome-like structures). We next tested the redifferentiation potential of 3D HDFs by exposing the disaggregated cells to endothelial growth conditions on Matrigel. When exposed to EGF and FGF-2, endothelial-like networks formed within 7 days, which was reduced to 24h following the addition of IGFI, VEGF and BMP4. These cells were also shown to express low levels of endothelial cell markers, VE-Cadherin, Nrp1, Nrp2 and ALK1 and higher levels of EphB4 and KDR by QPCR. These findings suggest that human cell differentiation can be efficiently reversed by physiological means and then re-differentiated into a new cell type