Population Of Pluripotent Cells Research Articles

WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus.

Plants can regenerate their bodies via de novo establishment of shoot apical meristems (SAMs) from pluripotent callus. Only a small fraction of callus cells is eventually specified into SAMs but the molecular mechanisms underlying fate specification remain obscure. The expression of WUSCHEL (WUS) is an early hallmark of SAM fate acquisition. Here, we show that a WUS paralog, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), negatively regulates SAM formation from callus in Arabidopsis thaliana. WOX13 promotes non-meristematic cell fate via transcriptional repression of WUS and other SAM regulators and activation of cell wall modifiers. Our Quartz-Seq2-based single cell transcriptome revealed that WOX13 plays key roles in determining cellular identity of callus cell population. We propose that reciprocal inhibition between WUS and WOX13 mediates critical cell fate determination in pluripotent cell population, which has a major impact on regeneration efficiency.

The Tip60/Ep400 chromatin remodeling complex impacts basic cellular functions in cranial neural crest-derived tissue during early orofacial development

The cranial neural crest plays a fundamental role in orofacial development and morphogenesis. Accordingly, mutations with impact on the cranial neural crest and its development lead to orofacial malformations such as cleft lip and palate. As a pluripotent and dynamic cell population, the cranial neural crest undergoes vast transcriptional and epigenomic alterations throughout the formation of facial structures pointing to an essential role of factors regulating chromatin state or transcription levels. Using CRISPR/Cas9-guided genome editing and conditional mutagenesis in the mouse, we here show that inactivation of Kat5 or Ep400 as the two essential enzymatic subunits of the Tip60/Ep400 chromatin remodeling complex severely affects carbohydrate and amino acid metabolism in cranial neural crest cells. The resulting decrease in protein synthesis, proliferation and survival leads to a drastic reduction of cranial neural crest cells early in fetal development and a loss of most facial structures in the absence of either protein. Following heterozygous loss of Kat5 in neural crest cells palatogenesis was impaired. These findings point to a decisive role of the Tip60/Ep400 chromatin remodeling complex in facial morphogenesis and lead us to conclude that the orofacial clefting observed in patients with heterozygous KAT5 missense mutations is at least in part due to disturbances in the cranial neural crest.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

BackgroundThere are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79–88, 2012) to answer these questions.ResultsWe identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis.ConclusionsCell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

Stem Cells Era in Maxillofacial Surgery

Stem cells provide a new era for oral and maxillofacial surgery and reconstruction. Stem cells are colonlogic cells that have the ability to renew themselves and form various predecessors. They are found in common tissues used for wound healing and recovery after injuries. Large numbers of these cells are known to be instantly accessible; no immunological denial or rejection; do not have host disease against graft; Tumorigenesis do not occur; but there are some small ethical problems; The predictable differentiation potential for tissue separation and integration is not surprising. There are two basic types of stem cells, depending on their origin: • Embryonic Stem Cells (ESCs): ESCs are initiated by a fetus that has been fertilized in an in vitro fertilization clinic (100,200 organized cell blastocysts). They are called a pluripotent cell population and are characterized by the ability to maintain the karyotype. ESCs can be divided into the three embryonic cotyledons (ectoderm, endoderm, and mesoderm); and in a culture; they form in colonies. These important highlights make ESCs affordable for future use in regenerative medicine and pharmaceuticals. Still; ethical discussions about the use of human embryos cannot be decided entirely; because in addition to scientific and practical questions, it is also dealt with through religious laws and philosophical reflections on the nature of human life. • Adult stem cells: Adult stem cells are found in fetal and postnatal tissues; They are more specialized (multipotent) cells that participate in the repair and restoration of tissues, as well as in the maturation and aging processes. There are numerous sources of adult stem cells such as bone marrow (BMMSC); Adipose tissue (ATMSCs); Liver; Umbilical cord; and muscles. The intraoral sources are the dental pulp; The periodontal ligaments can also be a source of adult stem cells. Fat and bone marrow stem cells are successful and are produced in the context of restorative work and aesthetics. However, ATMSCs have been less easily obtained without harmful effects on patients. Adipose tissue contains a population of mesenchymal stem cells that can be isolated and differentiated into various cell lines, including osteocytes; Fat cells; and myocytes depending on the culture conditions. These cells are called adipose derived stem cells (ASC). The question arises here is: what do stem cells do? And the answer is so simple when you know that 10 million cells in your body die every minute every day and your own stem cells replace them so you can continue living. Both undifferentiated ATMSC and BMMSC have osteogenic; chondrogenic and lipogenic differentiation potential. Both produce markers that help repair the deformity and have the same morphology; spindle shaped cells. Both appeared to work positively when used in reconstructive treatments. There are some contrasts between them; but the main imperative is; the ease of collection and the lower morbidity on the part of the donor. These two variables were extremely critical and favor the choice between fat and bone marrow for repair. ATMSCs and BMMSCs may be suitable replacements for the traditional intrusive method in the future. A better understanding of stem cell types, different platforms, and the influence of developmental components on the tissue design of different scaffolds, and how growth factors affect the tissue engineering pathway, opens opportunities to monitor recovery of bones and soft defects in the future, thus providing an alternative and innovative treatment for patients with soft / hard tissue defects.

Mitochondrial Effects on Seeds of Cancer Survival in Leukemia.

The cancer metabolic alteration is considered a hallmark and fast becoming a road for therapeutic intervention. Mitochondria have been regarded as essential cell elements that fuel the metabolic needs of most cancer cell types. Leukemia stem cells (LSCs) are a heterogeneous, highly self-renewing, and pluripotent cell population within leukemic cells. The most important source of ATP and metabolites to fulfill the bioenergetics and biosynthetic needs of most cancer stem cells is the mitochondria. In addition, mitochondria have a core role in autophagy and cell death and are the main source of reactive oxygen species (ROS) generation. Overall, growing evidence now shows that mitochondrial activities and pathways have changed to adapt with different types of leukemia, thus mitochondrial metabolism could be targeted for blood malignancy therapy. This review focuses on the function of mitochondria in LSC of the different leukemia types.

SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development.

ObjectivesGene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.Materials and methodsTo analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.ResultsOur results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.

Quantitative analysis of signaling responses during mouse primordial germ cell specification.

ABSTRACTDuring early mammalian development, the pluripotent cells of the embryo are exposed to a combination of signals that drive exit from pluripotency and germ layer differentiation. At the same time, a small population of pluripotent cells give rise to the primordial germ cells (PGCs), the precursors of the sperm and egg, which pass on heritable genetic information to the next generation. Despite the importance of PGCs, it remains unclear how they are first segregated from the soma, and if this involves distinct responses to their signaling environment. To investigate this question, we mapped BMP, MAPK and WNT signaling responses over time in PGCs and their surrounding niche in vitro and in vivo at single-cell resolution. We showed that, in the mouse embryo, early PGCs exhibit lower BMP and MAPK responses compared to neighboring extraembryonic mesoderm cells, suggesting the emergence of distinct signaling regulatory mechanisms in the germline versus soma. In contrast, PGCs and somatic cells responded comparably to WNT, indicating that this signal alone is not sufficient to promote somatic differentiation. Finally, we investigated the requirement of a BMP response for these cell fate decisions. We found that cell lines with a mutation in the BMP receptor (Bmpr1a−/−), which exhibit an impaired BMP signaling response, can efficiently generate PGC-like cells revealing that canonical BMP signaling is not cell autonomously required to direct PGC-like differentiation.

Fitness selection in human pluripotent stem cells and interspecies chimeras: Implications for human development and regenerative medicine

A small number of pluripotent cells within early embryo gives rise to all cells in the adult body, including germ cells. Hence, any mutations occurring in the pluripotent cell population are at risk of being propagated to their daughter cells and could lead to congenital defects or embryonic lethality and pose a risk of being transmitted to future generations. The observation that genetic errors are relatively common in preimplantation embryos, but their levels reduce as development progresses, suggests the existence of mechanisms for clearance of aberrant, unfit or damaged cells. Although early human embryogenesis is largely experimentally inaccessible, pluripotent stem cell (PSC) lines can be derived either from the inner cell mass (ICM) of a blastocyst or by reprogramming somatic cells into an embryonic stem cell-like state. PSCs retain the ability to differentiate into any cell type in vitro and, hence, they represent a unique and powerful tool for studying otherwise intractable stages of human development. The advent of PSCs has also opened up a possibility of developing regenerative medicine therapies, either through PSC differentiation in vitro or by creating interspecies chimeras for organ replacement. Here, we discuss the emerging evidence of cell selection in human PSC populations in vivo and in vitro and we highlight the implications of understanding this phenomenon for human development and regenerative medicine.

Роль транскрипционных факторов коктейля Яманаки (OCT4, sOX2, KLF4, c-Myc) в дифференцировке соматических клеток, их злокачественной трансформации и прогрессировании опухолей

The paper reviews the role of transcription factors that are part of the Yamanaka cocktail (OCT4, SOX2, KLF4, and c-Myc) in the mechanisms of tumor stem cell (TSC) differentiation (a pool of pluripotent cells), their participation in the malignant transformation of somatic cells, and in tumor progression. Their increased proliferative activity, which determines their malignant potential, was believed to be the main feature of tumor cells. Currently, there is more evidence supporting another concept that explains the continued tumor growth, metastasis, and resistance to therapy of most malignant tumors with a small, rest-ing, and rarely dividing population of pluripotent tumor cells. Year 2006 is considered to be the beginning of a new direction in developmental biology when Japanese researchers K. Takahashi and S. Yamanaka published the results of their studies on the properties of embryonic stem cells and experiments on direct reprogramming of somatic terminally differentiated cells.The research proved that four transcription factors were sufficient to maintain the pluripotent properties of cells, namely Oct4, SOX2, KLF4, and c-Myc, which were later called the Yamanaka cocktail. Such factors as SOX2and Oct4 are at the top of the hierarchy of transcription factors that regulate the pluripotent proper-ties of cells, their differentiation, and dedifferentiation. TSCs represent, albeit a complex, but very promising target for the development of innovative products for the diagnosis of and targeted therapy for neoplasms. From this point of view, the factors of pluripotency, which are parts of the Yamanaka cocktail, could be such promising targets, whose effect will probably be able to suppress or reduce the malignant potential of the most aggressive tumors and even prevent carcinogenic transformation in cases of precancerous pathology. Keywords: Yamanaka cocktail, OCT4, SOX2, KLF4, c-Myc, malignant transformation

Hematopoietic Stem Cells

The small population of pluripotent hematopoietic stem cells (PHSCs) in the bone marrow consists of short-term reconstituting cells (STRCs) and long-term reconstituting cells (LTRCs), based on how quickly the transplanted cells can produce progeny in an irradiated recipient. They can be “purified” using a combination of cell size; density; fluorescent dye uptake; resistance to cytotoxic chemicals; and cell-surface markers including Thy 1.1 (T), Sca-1 (S), c-kit (K), lineage (L), CD38 (38), and CD34 (34). Using five-color fluorescence-activated cell sorting the long term, very primitive mouse LTRCs are L-/lo, S+, K+, 38+, 34-, and appear to mature to L-/lo, S+, K+, 38+, 34+ cells and then to L-/lo, S+, K+, 38-, 34+; thus, STRCs acquire CD34 and lose CD38 on maturation from CD34- LTRCs. CD34 has been used to isolate PHSCs for human transplantation studies; therefore, the LTRC may be lost during this procedure. Experimental transplantation studies indicate that the best reconstitution occurs when both cell populations are present, the more mature cells activating the immature cells after myeloablation, whereas the mature cells provide negative control in normal animals. Functionally the type of assay that has been most widely used for the quantitation of mouse stem cells is the in vivo repopulating assay. Different numbers of donor cells are combined with a standard number of normal bone marrow cells. The normal cells protect against the immediate effects of myeloablation and compete with the donor stem cells. The proportions of mature cells derived from the donor stem cells are determined by the detection of a donor-specific marker, such as an isoenzyme, Y-chromosome, or congenic antigen. Similarly, using limiting dilution transplant of a donor test population of cells and a standard number of stem cell—compromised serially transplanted cells, the relative contribution of the donor cells is measured as a competitive repopulating unit. Finally, the repopulating stem cell unit assay using complete myeloablation and busulfan-treated bone marrow radioprotective support cells provides comparatively rapid and sensitive detection of the very small numbers of LTRCs present in limiting dilution transplants. This procedure utilizes busulfan because it appears preferentially stem cell toxic, and it provides radioprotective support cells that are unable to compete effectively with normal donor stem cells in the population under investigation. Stem cells are selected based on their ability to produce both lymphoid and myeloid repopulation in severely ablated mice, rather than competitive ability.

Mesenchymal stem cells reduce alcoholic hepatitis in mice via suppression of hepatic neutrophiland macrophageinfiltration,and of oxidative stress.

Mesenchymal stem cells (MSCs) are a population of pluripotent cells that have been tested for the treatment of many inflammatory diseases. It remains unclear whether MSCs were effective in treating mice with alcoholic hepatitis (AH) and its underlying mechanism. In the present study, MSCs were isolated from bone marrow of 4–6 week-old C57BL/6N male mice. AH was induced in female mice by chronic-binge ethanol feeding for 10 days. Intraperitoneal (i.p.) transplantation of MSCs or saline were performed in mice on day 10. Blood samples and hepatic tissues were harvested on day 11. Biochemical, liver histological and flow cytometric analyses were performed. Compared to the control mice, the AH mice had significantly increased liver/body weight ratio, serum alanine aminotransferase (ALT) and aspartate aminotransferases (AST), hepatic total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA), hepatic neutrophil and macrophage infiltration (P<0.001), which were markedly reduced by i.p. transplantation of MSCs (P<0.01). Compared to the control mice, the hepatic glutathione (GSH) was prominently lower in the AH mice (P<0.001), which was markedly enhanced after i.p. injection of MSCs (P<0.001). MSCs were effective for the treatment of AH mice, which might be associated with their ability in inhibiting hepatic neutrophil and macrophage infiltration, and alleviating oxidative stress.

Mesenchymal stem cells alleviate liver injury induced by chronic-binge ethanol feeding in mice via release of TSG6 and suppression of STAT3 activation

BackgroundMesenchymal stem cells (MSCs) are a population of pluripotent cells that might be used for treatment of liver disease. However, the efficacy of MSCs for mice with alcoholic hepatitis (AH) and its underlying mechanism remains unclear.MethodsMSCs were isolated from the bone marrow (BM) of 4–6-week-old male C57BL/6 N mice. AH was induced in female mice by chronic-binge ethanol feeding for 10 days. The mice were given intraperitoneal injections of MSCs with or without transfection or AG490, recombinant mouse tumor necrosis factor (TNF)-α-stimulated gene/protein 6 (rmTSG-6), or saline at day 10. Blood samples and hepatic tissues were collected at day 11. Various assays such as biochemistry, histology, and flow cytometry were performed.ResultsMSCs reduced AH in mice, decreasing liver/body weight ratio, liver injury, blood and hepatic lipids, malondialdehyde, interleukin (IL)-6, and TNF-ɑ, but increasing glutathione, IL-10, and TSG-6, compared to control mice. Few MSCs engrafted into the inflamed liver. Knockdown of TSG-6 in MSCs significantly attenuated their effects, and injection of rmTSG-6 achieved similar effects to MSCs. The signal transducer and activator of transcription 3 (STAT3) was activated in mice with AH, and MSCs and rmTSG-6 inhibited the STAT3 activation. Injection of MSCs plus AG490 obtained more alleviation of liver injury than MSCs alone.ConclusionsBM-MSCs injected into mice with AH do not engraft the liver, but they secrete TSG-6 to reduce liver injury and to inhibit STAT3 activation.

Epiblast Formation by TEAD-YAP-Dependent Expression of Pluripotency Factors and Competitive Elimination of Unspecified Cells

The epiblast is a pluripotent cell population first formed in preimplantation embryos, and its quality is important for proper development. Here, we examined the mechanisms of epiblast formation and found that the Hippo pathway transcription factor TEAD and its coactivator YAP regulate expression of pluripotency factors. After specification of the inner cell mass, YAP accumulates in the nuclei and activates TEAD. TEAD activity is required for strong expression of pluripotency factors and is variable in the forming epiblast. Cells showing low TEAD activity are eliminated from the epiblast through cell competition. Pluripotency factor expression and MYC control cell competition downstream of TEAD activity. Cell competition eliminates unspecified cells and is required for proper organization of the epiblast. These results suggest that induction of pluripotency factors by TEAD activity and elimination of unspecified cells via cell competition ensure the production of an epiblast with naive pluripotency.

Mesenchymal Stromal Cells Isolated from Irradiated Human Skin Have Diminished Capacity for Proliferation, Differentiation, Colony Formation, and Paracrine Stimulation.

Ionizing radiation, commonly used in the treatment of solid tumors, has unintended but deleterious effects on overlying skin and is associated with chronic nonhealing wounds. Skin‐derived mesenchymal stromal cells (SMSCs) are a pluripotent population of cells that are critically involved in skin homeostasis and wound healing. The aim of this study was to isolate and functionally characterize SMSCs from human skin that was previously irradiated as part of neoadjuvant or adjuvant cancer therapy. To this end, SMSCs were isolated from paired irradiated and nonirradiated human skin samples. Irradiated SMSCs expressed characteristic SMSC markers at lower levels, had disorganized cytoskeletal structure, and had disordered morphology. Functionally, these cells had diminished proliferative capacity and substantial defects in colony‐forming capacity and differentiation in vitro. These changes were associated with significant differential expression of genes known to be involved in skin physiology and wound healing. Conditioned media obtained from irradiated SMSCs affected fibroblast but not endothelial cell proliferation and migration. These results suggest that in situ damage to SMSCs during neoadjuvant or adjuvant radiation may play a critical role in the pathogenesis of slow or nonhealing radiation wounds. stem cells translational medicine 2019;8:925&934

Mesenchymal Stem Cells to Treat Digestive System Disorders: Progress Made and Future Directions

The purpose of this review is to summarize the results from clinical trials that evoked promise and discouragement for the use of mesenchymal stem cells (MSCs) to treat digestive system disorders. Adult MSCs are defined as a non-homogeneous population of pluripotent progenitor cells, which can be isolated and expanded in vitro from different tissues. The differentiation capacity of MSC along mesenchymal lineages and their immunomodulatory properties have been considered a new therapeutic approach for intestinal disorders. A dysregulated immune response is the cause and sustainment of these disorders, as they are characterized by progressive tissue damage with no available curative treatment. Up to now, 130 clinical trials on MSC-based therapy are registered to treat conditions affecting the digestive system. The results from completed or underway clinical studies are encouraging, showing both benefit for those digestive disorders refractory to any conventional therapy and progression toward end-stage liver disease. However, the absence of large, robust controlled, and randomized clinical trials to assess MSC clinical efficacy limits MSC-based therapy translation to bedside reality to completely cure digestive disorders.

HDAC6 differentially regulates autophagy in stem-like versus differentiated cancer cells

ABSTRACTCancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

The epigenetic modifier Fam208a is required to maintain epiblast cell fitness

Gastrulation initiates with the formation of the primitive streak, during which, cells of the epiblast delaminate to form the mesoderm and definitive endoderm. At this stage, the pluripotent cell population of the epiblast undergoes very rapid proliferation and extensive epigenetic programming. Here we show that Fam208a, a new epigenetic modifier, is essential for early post-implantation development. We show that Fam208a mutation leads to impaired primitive streak elongation and delayed epithelial-to-mesenchymal transition. Fam208a mutant epiblasts had increased expression of p53 pathway genes as well as several pluripotency-associated long non-coding RNAs. Fam208a mutants exhibited an increase in p53-driven apoptosis and complete removal of p53 could partially rescue their gastrulation block. This data demonstrates a new in vivo function of Fam208a in maintaining epiblast fitness, establishing it as an important factor at the onset of gastrulation when cells are exiting pluripotency.

Evaluating Synthetic Activation and Repression of Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and Astrocytes.

SummaryModulation of transcription, either synthetic activation or repression, via dCas9-fusion proteins is a relatively new methodology with the potential to facilitate high-throughput up- or downregulation studies of gene function. Genetic studies of neurodevelopmental disorders have identified a growing list of risk variants, including both common single-nucleotide variants and rare copy-number variations, many of which are associated with genes having limited functional annotations. By applying a CRISPR-mediated gene-activation/repression platform to populations of human-induced pluripotent stem cell-derived neural progenitor cells, neurons, and astrocytes, we demonstrate that it is possible to manipulate endogenous expression levels of candidate neuropsychiatric risk genes across these three cell types. Although proof-of-concept studies using catalytically inactive Cas9-fusion proteins to modulate transcription have been reported, here we present a detailed survey of the reproducibility of gRNA positional effects across a variety of neurodevelopmental disorder-relevant risk genes, donors, neural cell types, and dCas9 effectors.

Dual roles of Akirin2 protein during Xenopus neural development

To ensure correct spatial and temporal patterning, embryos must maintain pluripotent cell populations and control when cells undergo commitment. The newly identified nucleoprotein Akirin has been shown to modulate the innate immune response through epigenetic regulation and to play important roles in other physiological processes, but its role in neural development remains unknown. Here we show that Akirin2 is required for neural development in Xenopus and that knockdown of Akirin2 expands the expression of the neural progenitor marker Sox2 and inhibits expression of the differentiated neuronal marker N-tubulin. Akirin2 acts antagonistically to Geminin, thus regulating Sox2 expression, and maintains the neural precursor state by participating in the Brg1/Brm-associated factor (BAF) complex mediated by BAF53a. Additionally, Akirin2 also modulates N-tubulin expression by acting upstream of neuronal differentiation 1 (NeuroD) and in parallel with neurogenin-related 1 (Ngnr1) during terminal neuronal differentiation. Thus, our results reveal a novel model in which Akirin2 precisely coordinates and temporally controls Xenopus neural development.