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.

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.

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.

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.

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).

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.

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.

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.

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).

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6β1 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 α6β1 and inactivation of focal adhesion kinase (FAK).

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.

Derivation and long-term culture of transgene-free human induced pluripotent stem cells on synthetic substrates.

We describe a platform to derive, culture, and differentiate genomically stable, transgene-free human induced pluripotent stem cells (iPSCs) on a fully synthetic polymer substrate made of a grafted zwitterionic hydrogel: poly2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl) ammonium hydroxide (PMEDSAH). Three independent transgene-free iPSC lines derived in these conditions demonstrated continuous self-renewal, genomic stability, and pluripotency in vitro and in vivo after up to 9 months of continuous in vitro culture on PMEDSAH-grafted plates. Together, these data demonstrate the strength this alternative platform offers to generate and maintain human iPSCs for regenerative medicine.

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.

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.

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.

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.

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.

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.

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.

Synthetic polymer coatings for long-term growth of human embryonic stem cells.

We report a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), which sustains long-term human embryonic stem (hES) cell growth in several different culture media, including commercially available defined media. The development of a standardized, controllable and sustainable culture matrix for hES cells is an essential step in elucidating mechanisms that control hES cell behavior and in optimizing conditions for biomedical applications 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.

Microfluidic culture of single human embryonic stem cell colonies.

We have developed a miniaturized microfluidic culture system that allows experimentation on individual human embryonic stem cell (hESC) colonies in dynamic (flow applied) or static (without flow) conditions. The system consists of three inlet channels that converge into a cell-culture channel and provides the capability to spatially and temporally deliver specific treatments by using patterned laminar fluid flow to different parts of a single hESC colony. We show that microfluidic culture for 96 h with or without flow results in similar maintenance of hESC self-renewal, the capability to differentiate into three germ cell lineages, and to maintain a normal karyotype, as in standard culture dishes.

Analysis of the factors that limit the ability of feeder cells to maintain the undifferentiated state of human embryonic stem cells.

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved.

Activation of p38 MAPK during porcine oocyte maturation.

The p38 MAPK is a member of the mitogen-activated protein kinase (MAPK) family that participates in a signaling cascade in response to cytokines and stress in somatic cells. The present study was designed to investigate the expression and possible function of p38 MAPK in porcine oocytes during maturation. In immunoblots, p38 MAPK was detected in oocytes and cumulus cells.