Selective Inhibition of mTORC1 Signaling Supports the Development and Maintenance of Pluripotency.

Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.

Intratumoural haematopoietic stem and progenitor cell differentiation into M2 macrophages facilitates the regrowth of solid tumours after radiation therapy.

BACKGROUND: Bone-marrow-derived haematopoietic stem and progenitor cells (HSPCs) are a prominent part of the highly complex tumour microenvironment (TME) where they localise within tumours and maintain haematopoietic potency. Understanding the role HSPCs play in tumour growth and response to radiation therapy (RT) may lead to improved patient treatments and outcomes. METHODS: We used a mouse model of non-small cell lung carcinoma where tumours were exposed to RT regimens alone or in combination with GW2580, a pharmacological inhibitor of colony stimulating factor (CSF)-1 receptor. RT-PCR, western blotting and immunohistochemistry were used to quantify expression levels of factors that affect HSPC differentiation. DsRed+ HSPC intratumoural activity was tracked using flow cytometry and confocal microscopy. RESULTS: We demonstrated that CSF-1 is enhanced in the TME following exposure to RT. CSF-1 signaling induced intratumoural HSPC differentiation into M2 polarised tumour-associated macrophages (TAMs), aiding in post-RT tumour survival and regrowth. In contrast, hyperfractionated/pulsed radiation therapy (PRT) and GW2580 ablated this process resulting in improved tumour killing and mouse survival. CONCLUSIONS: Tumours coopt intratumoural HSPC fate determination via CSF-1 signaling to overcome the effects of RT. Thus, limiting intratumoural HSPC activity represents an attractive strategy for improving the clinical treatment of solid tumours.

Identification of biomarkers indicative of functional skeletal stem cells.

OBJECTIVES: Skeletal stem cells (SSCs) are characterized by expression of cell surface biomarkers and their ability to differentiate into bone, cartilage and fat. However, the current biomarkers used to identify these cell populations are not cell-type-specific or indicative of the differentiation status of these cells and are therefore unreliable. Our objective was to identify alternative cell surface biomarkers and transcription factors shared between SSCs isolated from the bone marrow (BM) and those derived from pluripotent stem cells (PSC). MATERIALS AND METHODS: Human PSCs were induced into SSCs. FACS and qRT-PCR were used to determine differences in expression of cell surface biomarkers and transcription factors between SSCs derived from PSCs and isolated from BM, in differentiating cells, in cells from early and late passage, and in fibroblasts. RESULTS: A significant reduction in proliferation and capacity of SSCs to differentiate into adipocytes and osteoblasts was observed after 3 passages. Protein and mRNA analysis indicated that commonly used biomarkers remain highly expressed in cells that lost capacity for differentiation. However, integrin α6 (CD49f) and transcription factors GATA6, PRDM16, SIM2 and SOX11 were significantly upregulated in SSCs compared to fibroblasts. In early stages of adipogenic and osteogenic differentiation, the expression of CD49f, GATA6 and SIM2 was reduced in later passage cells, which have limited proliferation and differentiation capabilities. CONCLUSIONS: Our results suggest that CD49f and transcription factors GATA6 and SIM2 identify functional SSCs.

DPPA5 Supports Pluripotency and Reprogramming by Regulating NANOG Turnover.

Although a specific group of transcription factors such as OCT4, SOX2, and NANOG are known to play essential roles in pluripotent stem cell (PSC) self-renewal, pluripotency, and reprogramming, other factors and the key signaling pathways regulating these important properties are not completely understood. Here, we demonstrate that the PSC marker Developmental Pluripotency Associated 5 (DPPA5) plays an important role in human PSC (hPSC) self-renewal and cell reprogramming in feeder-free conditions. Compared to hPSCs grown on mouse embryonic fibroblasts, cells cultured on feeder-free substrates, such as Matrigel, Laminin-511, Vitronectin, or the synthetic polymer poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide], had significantly higher DPPA5 gene expression and protein levels. Overexpression of DPPA5 in hPSCs increased NANOG protein levels via a post-transcriptional mechanism. Coimmunoprecipitation, protein stability assays, and quantitative RT-PCR, demonstrated that DPPA5 directly interacted, stabilized, and enhanced the function of NANOG in hPSCs. Additionally, DPPA5 increased the reprogramming efficiency of human somatic cells to induced pluripotent stem cells (hiPSCs). Our study provides new insight into the function of DPPA5 and NANOG regulation in hPSCs.

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6ß1 Supports Human Pluripotent Stem Cell Self-Renewal.

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6ß1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6ß1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of ß1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin ß1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6ß1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6ß1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764.

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells.

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons derived from hPSCs within 23 days of culture using soft PMAs were improved more than fourfold and tenfold, respectively, compared with coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo/YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Enhanced Expansion of Human Pluripotent Stem Cells and Somatic Cell Reprogramming Using Defined and Xeno-Free Culture Conditions.

Human embryonic stem cells and induced pluripotent stem cells (hPSC) have an unprecedented opportunity to revolutionize the fields of developmental biology as well as tissue engineering and regenerative medicine. However, their applications have been significantly limited by the lack of chemically defined and xeno-free culture conditions. The demand for the high-quality and scaled-up production of cells for use in both research and clinical studies underscores the need to develop tools that will simplify the in vitro culture process while reducing the variables. Here, we describe a systematic study to identify the optimal conditions for the initial cell attachment of hPSC to tissue culture dishes grafted with polymers of N-(3-Sulfopropyl)-N-Methacryloxyethyl-N, N-Dimethylammoniun Betaine (PMEDSAH) in combination with chemically defined and xeno-free culture media. After testing multiple supplements and chemicals, we identified that pre-conditioning of PMEDSAH grafted plates with 10% human serum (HS) supported the initial cell attachment, which allowed for the long-term culture and maintenance of hPSC compared to cells cultured on Matrigel-coated plates. Using this culture condition, a 2.1-fold increase in the expansion of hPSC was observed without chromosomal abnormalities. Furthermore, this culture condition supported a higher reprogramming efficiency (0.37% vs. 0.22%; p < 0.0068) of somatic cells into induced pluripotent stem cells compared to the non-defined culture conditions. This defined and xeno-free hPSC culture condition may be used in obtaining the large populations of hPSC and patient-derived iPSC required for many applications in regenerative and translational medicine.

Regulation and Functions of α6-Integrin (CD49f) in Cancer Biology.

Over the past decades, our knowledge of integrins has evolved from being understood as simple cell surface adhesion molecules to receptors that have a complex range of intracellular and extracellular functions, such as delivering chemical and mechanical signals to cells. Consequently, they actively control cellular proliferation, differentiation, and apoptosis. Dysregulation of integrin signaling is a major factor in the development and progression of many tumors. Many reviews have covered the broader integrin family in molecular and cellular studies and its roles in diseases. Nevertheless, further understanding of the mechanisms specific to an individual subunit of different heterodimers is more useful. Thus, we describe the current understanding of and exploratory investigations on the α6-integrin subunit (CD49f, VLA6; encoded by the gene itga6) in normal and cancer cells. The roles of ITGA6 in cell adhesion, stemness, metastasis, angiogenesis, and drug resistance, and as a diagnosis biomarker, are discussed. The role of ITGA6 differs based on several features, such as cell background, cancer type, and post-transcriptional alterations. In addition, exosomal ITGA6 also implies metastatic organotropism. The importance of ITGA6 in the progression of a number of cancers, including hematological malignancies, suggests its potential usage as a novel prognostic or diagnostic marker and useful therapeutic target for better clinical outcomes.

Gene therapy: implications for craniofacial regeneration.

Gene therapy in the craniofacial region provides a unique tool for delivery of DNA to coordinate protein production in both time and space. The drive to bring this technology to the clinic is derived from the fact that more than 85% of the global population may at one time require repair or replacement of a craniofacial structure. This need ranges from mild tooth decay and tooth loss to temporomandibular joint disorders and large-scale reconstructive surgery. Our ability to insert foreign DNA into a host cell has been developing since the early uses of gene therapy to alter bacterial properties for waste cleanup in the 1980s followed by successful human clinical trials in the 1990s to treat severe combined immunodeficiency. In the past 20 years, the emerging field of craniofacial tissue engineering has adopted these techniques to enhance regeneration of mineralized tissues, salivary gland, and periodontium and to reduce tumor burden of head and neck squamous cell carcinoma. Studies are currently pursuing research on both biomaterial-mediated gene delivery and more clinically efficacious, although potentially more hazardous, viral methods. Although hundreds of gene therapy clinical trials have taken place in the past 20 years, we must still work to ensure an ideal safety profile for each gene and delivery method combination. With adequate genotoxicity testing, we can expect gene therapy to augment protein delivery strategies and potentially allow for tissue-specific targeting, delivery of multiple signals, and increased spatial and temporal control with the goal of natural tissue replacement in the craniofacial complex.

Progress in the Use of Induced Pluripotent Stem Cell-Derived Neural Cells for Traumatic Spinal Cord Injuries in Animal Populations: Meta-Analysis and Review.

Induced pluripotent stem cells (iPSCs) are cells genetically reprogrammed from somatic cells, which can be differentiated into neurological lineages with the aim to replace or assist damaged neurons in the treatment of spinal cord injuries (SCIs) caused by physical trauma. Here, we review studies addressing the functional use of iPSC-derived neural cells in SCIs and perform a meta-analysis to determine if significant motor improvement is restored after treatment with iPSC-derived neural cells compared with treatments using embryonic stem cell (ESC)-derived counterpart cells and control treatments. Overall, based on locomotion scales in rodents and monkeys, our meta-analysis indicates a therapeutic benefit for SCI treatment using neural cells derived from either iPSCs or ESCs, being this of importance due to existing ethical and immunological complications using ESCs. Results from these studies are evidence of the successes and limitations of iPSC-derived neural cells in the recovery of motor capacity. Stem Cells Translational Medicine 2019;8:681&693.

Integrin α6 (CD49f), The Microenvironment and Cancer Stem Cells.

Cancer is a highly prevalent and potentially terminal disease that affects millions of individuals worldwide. Here, we review the literature exploring the intricacies of stem cells bearing tumorigenic characteristics and collect evidence demonstrating the importance of integrin α6 (ITGA6, also known as CD49f) in cancer stem cell (CSC) activity. ITGA6 is commonly used to identify CSC populations in various tissues and plays an important role sustaining the self-renewal of CSCs by interconnecting them with the tumorigenic microenvironment.

Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.

Nanotopography regulates motor neuron differentiation of human pluripotent stem cells.

The regulation of human pluripotent stem cell (hPSC) behaviors has been mainly studied through exploration of biochemical factors. However, the current directed differentiation protocols for hPSCs that completely rely on biochemical factors remain suboptimal. It has recently become evident that coexisting biophysical signals in the stem cell microenvironment, including nanotopographic cues, can provide potent regulatory signals to mediate adult stem cell behaviors, including self-renewal and differentiation. Herein, we utilized a recently developed, large-scale nanofabrication technique based on reactive-ion etching (RIE) to generate random nanoscale structures on glass surfaces with high precision and reproducibility. We report here that hPSCs are sensitive to nanotopographic cues and such nanotopographic sensitivity can be leveraged for improving directed neuronal differentiation of hPSCs. We demonstrate early neuroepithelial conversion and motor neuron (MN) progenitor differentiation of hPSCs can be promoted using nanoengineered topographic substrates. We further explore how hPSCs sense the substrate nanotopography and relay this biophysical signal through a regulatory signaling network involving cell adhesion, the actomyosin cytoskeleton, and Hippo/YAP signaling to mediate the neuroepithelial induction of hPSCs. Our study provides an efficient method for large-scale production of MNs from hPSCs, useful for regenerative medicine and cell-based therapies.

The Role of Integrin α6 (CD49f) in Stem Cells: More than a Conserved Biomarker.

Stem cells have the capacity for self-renewal and differentiation into specialized cells that form and repopulated all tissues and organs, from conception to adult life. Depending on their capacity for differentiation, stem cells are classified as totipotent (ie, zygote), pluripotent (ie, embryonic stem cells), multipotent (ie, neuronal stem cells, hematopoietic stem cells, epithelial stem cells, etc.), and unipotent (ie, spermatogonial stem cells). Adult or tissue-specific stem cells reside in specific niches located in, or nearby, their organ or tissue of origin. There, they have microenvironmental support to remain quiescent, to proliferate as undifferentiated cells (self-renewal), and to differentiate into progenitors or terminally differentiated cells that migrate from the niche to perform specialized functions. The presence of proteins at the cell surface is often used to identify, classify, and isolate stem cells. Among the diverse groups of cell surface proteins used for these purposes, integrin α6, also known as CD49f, may be the only biomarker commonly found in more than 30 different populations of stem cells, including some cancer stem cells. This broad expression among stem cell populations indicates that integrin α6 may play an important and conserved role in stem cell biology, which is reaffirmed by recent demonstrations of its role maintaining self-renewal of pluripotent stem cells and breast and glioblastoma cancer stem cells. Therefore, this review intends to highlight and synthesize new findings on the importance of integrin α6 in stem cell biology.

Enhancement of the propagation of human embryonic stem cells by modifications in the gel architecture of PMEDSAH polymer coatings.

Well-defined culture conditions are essential for realizing the full potential of human embryonic stem cells (hESCs) in regenerative medicine where large numbers of cells are required. Synthetic polymers such as poly[2-(methacryloyloxy) ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide] (PMEDSAH), offer multiple advantages over mouse embryonic fibroblasts (MEFs) and Matrigel™ for hESC culture and expansion. However, there is limited understanding of the mechanisms by which hESCs are propagated on synthetic polymers coatings. Here, the effects of PMEDSAH gel architecture on hESC self-renewal were determined. By increasing the atom transfer radical polymerization (ATRP) reaction time, the thickness of PMEDSAH was increased and its internal hydrogel architecture was modified, while maintaining its overall chemical structure. A 105 nm thick ATRP PMEDSAH coating showed a significant increase in the expansion rate of hESCs. Theoretical calculations suggested that 20,000 hESCs cultured on this substrate could be expanded up to 4.7 × 10(9) undifferentiated cells in five weeks. In addition, hESCs grown on ATRP PMEDSAH coatings retained pluripotency and displayed a normal karyotype after long-term culture. These data demonstrate the importance of polymer physical properties in hESC expansion. This modification of PMEDSAH coatings may be used to obtain large populations of hESCs required for many applications in regenerative medicine.

Human transgene-free amniotic-fluid-derived induced pluripotent stem cells for autologous cell therapy.

The establishment of a reliable prenatal source of autologous, transgene-free progenitor cells has enormous potential in the development of regenerative-medicine-based therapies for infants born with devastating birth defects. Here, we show that a largely CD117-negative population of human amniotic fluid mesenchymal stromal cells (AF-MSCs) obtained from fetuses with or without prenatally diagnosed anomalies are readily abundant and have limited baseline differentiation potential when compared with bone-marrow-derived MSCs and other somatic cell types. Nonetheless, the AF-MSCs could be easily reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai viral vectors encoding for OCT4, SOX2, KLF4, and cMYC. The iPSCs were virtually indistinguishable from human embryonic stem cells in multiple assays and could be used to generate a relatively homogeneous population of neural progenitors, expressing PAX6, SOX2, SOX3, Musashi-1, and PSA-NCAM, for potential use in neurologic diseases. Further, these neural progenitors showed engraftment potential in vivo and were capable of differentiating into mature neurons and astrocytes in vitro. This study demonstrates the usefulness of AF-MSCs as an excellent source for the generation of human transgene-free iPSCs ideally suited for autologous perinatal regenerative medicine applications.

Mechanics regulates fate decisions of human embryonic stem cells.

Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.

Nanotopography influences adhesion, spreading, and self-renewal of human embryonic stem cells.

Human embryonic stem cells (hESCs) have great potentials for future cell-based therapeutics. However, their mechanosensitivity to biophysical signals from the cellular microenvironment is not well characterized. Here we introduced an effective microfabrication strategy for accurate control and patterning of nanoroughness on glass surfaces. Our results demonstrated that nanotopography could provide a potent regulatory signal over different hESC behaviors, including cell morphology, adhesion, proliferation, clonal expansion, and self-renewal. Our results indicated that topological sensing of hESCs might include feedback regulation involving mechanosensory integrin-mediated cell-matrix adhesion, myosin II, and E-cadherin. Our results also demonstrated that cellular responses to nanotopography were cell-type specific, and as such, we could generate a spatially segregated coculture system for hESCs and NIH/3T3 fibroblasts using patterned nanorough glass surfaces.

Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells.

The culture of human embryonic stem (hES) cells in defined and xenogeneic-free conditions will contribute substantially to future biotechnological and medical applications. To achieve this goal, we developed the first fully defined synthetic polymer coating poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) that sustains long-term growth of hES cells in different culture media. Here we describe a detailed protocol for the reproducible fabrication of PMEDSAH coating on tissue culture polystyrene dishes, and for the feeder-free culture of hES cells on PMEDSAH coating in defined culture medium. This culture system represents a key step toward the fully defined and xenogeneic-free culture of hES cells.

Enhanced transfection efficiency of human embryonic stem cells by the incorporation of DNA liposomes in extracellular matrix.

Because human embryonic stem (hES) cells can differentiate into virtually any cell type in the human body, these cells hold promise for regenerative medicine. The genetic manipulation of hES cells will enhance our understanding of genes involved in early development and will accelerate their potential use and application for regenerative medicine. The objective of this study was to increase the transfection efficiency of plasmid DNA into hES cells by modifying a standard reverse transfection (RT) protocol of lipofection. We hypothesized that immobilization of plasmid DNA in extracellular matrix would be a more efficient method for plasmid transfer due to the affinity of hES cells for substrates such as Matrigel and to the prolonged exposure of cells to plasmid DNA. Our results demonstrate that this modification doubled the transfection efficiency of hES cells and the generation of clonal cell lines containing a piece of foreign DNA stably inserted in their genomes compared to results obtained with standard forward transfection. In addition, treatment with dimethyl sulfoxide further increased the transfection efficiency of hES cells. In conclusion, modifications to the RT protocol of lipofection result in a significant and robust increase in the transfection efficiency of hES cells.