PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.
View Article and Find Full Text PDFTankyrase/PARP inhibitor-regulated naïve human pluripotent stem cells (TIRN-hPSC) represent a new class of human stem cells for regenerative medicine that can differentiate into multi-lineage progenitors with improved in vivo functionality. Chemical reversion of conventional, primed hPSC to a TIRN-hPSC state alleviates dysfunctional epigenetic donor cell memory, lineage-primed gene expression, and potentially disease-associated aberrations in their differentiated progeny. Here, we provide methods for the reversion of normal or diseased patient-specific primed hPSC to TIRN-hPSC and describe their subsequent differentiation into embryonic-like pericytic-endothelial "naïve" vascular progenitors (N-VP).
View Article and Find Full Text PDFHuman pluripotent stem cells (hPSCs) can generate specialized cell lineages that have great potential for regenerative therapies and disease modeling. However, the developmental stage of the lineages generated from conventional hPSC cultures in vitro are embryonic in phenotype, and may not possess the cellular maturity necessary for corrective regenerative function in vivo in adult recipients. Here, we present the scientific evidence for how adult human tissues could generate human-animal interspecific chimeras to solve this problem.
View Article and Find Full Text PDFTankyrase 1 (TNKS1; PARP-5a) and Tankyrase 2 (TNKS2; PARP-5b) are poly-ADP-ribosyl-polymerase (PARP)-domain-containing proteins that regulate the activities of a wide repertoire of target proteins via post-translational addition of poly-ADP-ribose polymers (PARylation). Although tankyrases were first identified as regulators of human telomere elongation, important and expansive roles of tankyrase activity have recently emerged in the development and maintenance of stem cell states. Herein, we summarize the current state of knowledge of the various tankyrase-mediated activities that may promote human naïve and 'extended' pluripotency'.
View Article and Find Full Text PDFHere, we report that the functionality of vascular progenitors (VP) generated from normal and disease-primed conventional human induced pluripotent stem cells (hiPSC) can be significantly improved by reversion to a tankyrase inhibitor-regulated human naïve epiblast-like pluripotent state. Naïve diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve diabetic hiPSC (N-DhiPSC) possessed higher vascular functionality, maintained greater genomic stability, harbored decreased lineage-primed gene expression, and were more efficient in migrating to and re-vascularizing the deep neural layers of the ischemic retina than isogenic diabetic vascular progenitors (DVP). These findings suggest that reprogramming to a stable naïve human pluripotent stem cell state may effectively erase dysfunctional epigenetic donor cell memory or disease-associated aberrations in patient-specific hiPSC.
View Article and Find Full Text PDFNaïve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a naïve-like pluripotency with improved functionality. This chemical naïve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3β, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i).
View Article and Find Full Text PDFAlthough human embryonic stem cells (hESCs) were first derived almost 20 years ago, it was only recently acknowledged that they share closer molecular and functional identity to postimplantation lineage-primed murine epiblast stem cells than to naïve preimplantation inner cell mass-derived mouse ESCs (mESCs). A myriad of transcriptional, epigenetic, biochemical, and metabolic attributes have now been described that distinguish naïve and primed pluripotent states in both rodents and humans. Conventional hESCs and human induced pluripotent stem cells (hiPSCs) appear to lack many of the defining hallmarks of naïve mESCs.
View Article and Find Full Text PDFThe derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i).
View Article and Find Full Text PDFThe cancer stem cell (CSC) paradigm presumes the existence of self-renewing cancer cells capable of regenerating all tumor compartments and exhibiting stem cell-associated phenotypes. Recent interpretations of the CSC hypothesis envision stemness as a dynamic trait of tumor-initiating cells rather than a defined and unique cell type. Bidirectional crosstalk between the tumor microenvironment and the cancer bulk is well described in the literature and the tumor-associated stroma, vasculature and immune infiltrate have all been implicated as direct contributors to tumor development.
View Article and Find Full Text PDFBackground: The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells.
View Article and Find Full Text PDFMesenchymal stem/stromal cells (MSCs) and MSC-like multipotent stem/progenitor cells have been widely investigated for regenerative medicine and deemed promising in clinical applications. In order to further improve MSC-based stem cell therapeutics, it is important to understand the cellular kinetics and functional roles of MSCs in the dynamic regenerative processes. However, due to the heterogeneous nature of typical MSC cultures, their native identity and anatomical localization in the body have remained unclear, making it difficult to decipher the existence of distinct cell subsets within the MSC entity.
View Article and Find Full Text PDFCancer treatment generally relies on tumor ablative techniques that can lead to major functional or disfiguring defects. These post-therapy impairments require the development of safe regenerative therapy strategies during cancer remission. Many current tissue repair approaches exploit paracrine (immunomodulatory, pro-angiogenic, anti-apoptotic and pro-survival effects) or restoring (functional or structural tissue repair) properties of mesenchymal stem/stromal cells (MSC).
View Article and Find Full Text PDFWe have previously described the expression of CD44, CD90, CD117 and CD133 in NSCLC tumors, adjacent normal lung, and malignant pleural effusions (MPE). Here we describe the unique subset of tumors expressing CD117 (KIT), a potential therapeutic target. Tumor and adjacent tissue were collected from 58 patients.
View Article and Find Full Text PDFBackground Aims: Adipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system.
View Article and Find Full Text PDFThe stromal-vascular fraction (SVF) of adipose tissue is a rich source of multipotent stem cells. We and others have described three major populations of stem/progenitor cells in this fraction, all closely associated with small blood vessels: endothelial progenitor cells (EPC, CD45-/CD31+/CD34+), pericytes (CD45-/CD31-/CD146+), and supra-adventitial adipose stromal cells (SA-ASC, CD45-/CD31-/CD146-/CD34+). EPC are luminal, pericytes are adventitial, and SA-ASC surround the vessel like a sheath.
View Article and Find Full Text PDFNonviral conversion of skin or blood cells into clinically useful human induced pluripotent stem cells (hiPSC) occurs in only rare fractions (~0.001%-0.5%) of donor cells transfected with non-integrating reprogramming factors.
View Article and Find Full Text PDFThe hematopoietic and vascular lineages are intimately entwined as they arise together from bipotent hemangioblasts and hemogenic endothelial precursors during human embryonic development. In vitro differentiation of human pluripotent stem cells toward these lineages provides opportunities for elucidating the mechanisms of hematopoietic genesis. We previously demonstrated the stepwise in vitro differentiation of human embryonic stem cells (hESC) to definitive erythromyelopoiesis through clonogenic bipotent primitive hemangioblasts.
View Article and Find Full Text PDFBackground Aims: Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.
Methods: We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment.
Background: Adipose stem cells represent a heterogenous population. Understanding the functional characteristics of subpopulations will be useful in developing adipose stem cell-based therapies for regenerative medicine applications. The aim of this study was to define distinct populations within the stromal vascular fraction based on surface marker expression, and to evaluate the ability of each cell type to differentiate to mature adipocytes.
View Article and Find Full Text PDFOne of the major strengths of Flow Cytometry is its ability to perform multiple measurements on single cells within a heterogeneous mixture. When the populations of interest are relatively rare, analytical methodology that is adequate for more prevalent populations is often overcome by sources of artifacts that become apparent only when large numbers of cells are acquired. This chapter presents three practical examples of rare event problems and gives detailed instructions for preparation of single cell suspensions from bone marrow, malignant effusions, and solid tissue.
View Article and Find Full Text PDFPerivascular multipotent mesenchymal progenitors exist in a variety of tissues, including the placenta. Here, we suggest that the abundant vasculature present in the human placenta can serve as a source of myogenic cells to regenerate skeletal muscle. Chorionic villi dissected from the mid-gestation human placenta were first transplanted intact into the gastrocnemius muscles of SCID/mdx mice, where they participated in muscle regeneration by producing myofibers expressing human dystrophin and spectrin.
View Article and Find Full Text PDFThere is often a pressing need for reconstruction after cancer surgery. Regenerative therapy holds the promise of more natural and esthetic functional tissue. In the case of breast reconstruction postmastectomy, volume retention problems associated with autologous fat transfer could be ameliorated by augmentation with cells capable mediating rapid vascularization of the graft.
View Article and Find Full Text PDFAdipose-derived stem cells (ASCs) have been proposed to stabilize autologous fat grafts for regenerative therapy, but their safety is unknown in the setting of reconstructive surgery after mastectomy. Both bone marrow mesenchymal stem cells (MSCs) and ASC have been shown to enhance tumorigenesis of established breast cancer cell lines, but primary patient material has not been tested. Here, we ask whether ASC promote the in vitro growth and in vivo tumorigenesis of metastatic breast cancer clinical isolates.
View Article and Find Full Text PDFBackground: A variety of markers have been proposed to identify breast cancer stem cells. Here, we used immunohistostaining and flow cytometry to analyze their interrelationships and to sort cells for tumorigenicity studies.
Methods: Cytokeratin, CD44, and CD90 were localized to primary breast cancer and normal breast (NB) tissue by immunohistostaining and related to CD117 and CD133 expression by flow cytometry.