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Advances in cell-based therapy, particularly CAR-T cell therapy, have transformed the treatment of hematological malignancies. Although an important step forward for the field, autologous CAR-T therapies are hindered by high costs, manufacturing challenges, and limited efficacy against solid tumors. With ongoing progress in gene editing and culture techniques, engineered stem cells and their application in cell therapy are poised to address some of these challenges. Here, we review stem cell-based immunotherapy approaches, stem cell sources, gene engineering and manufacturing strategies, therapeutic platforms, and clinical trials, as well as challenges and future directions for the field.

Mouse embryonic stem cells (ESCs) require transcriptional regulation to ensure rapid proliferation that allows for self-renewal. However, the molecular mechanism by which transcriptional factors regulate this rapid proliferation remains largely unknown. Here we present data showing that CIBZ, a BTB domain zinc finger transcriptional factor, is a key transcriptional regulator for regulation of ESC proliferation. Here we show that deletion or siRNA knockdown of CIBZ inhibits ESC proliferation. Cell cycle analysis shows that loss of CIBZ delays the progression of ESCs through the G1 to S phase transition. Conversely, constitutive ectopic expression of exogenous CIBZ in ESCs promotes proliferation and accelerates G1/S transition. These findings suggest that regulation of the G1/S transition explains, in part, CIBZ-associated ESC proliferation. Our data suggest that CIBZ acts through the post-transcriptionally regulates the expression of Nanog, a positive regulator of ESC proliferation and G1/S transition, but does not affect Oct3/4 and Sox2 protein expression. Notably, constitutive overexpression of Nanog partially rescued the proliferation defect caused by CIBZ knockdown, indicating the role of CIBZ in ESC proliferation and G1/S transition at least in part depends on the Nanog protein level.

More research is being conducted on myocardial cell treatments utilizing stem cell lines that can develop into cardiomyocytes. All of the forms of cardiac illnesses have shown to be quite amenable to treatments using embryonic (ESCs) and induced pluripotent stem cells (iPSCs). In the present study, we reviewed the differentiation of these cell types into cardiomyocytes from an epigenetic standpoint. We also provided a miRNA network that is devoted to the epigenetic commitment of stem cells toward cardiomyocyte cells and related diseases, such as congenital heart defects, comprehensively. Histone acetylation, methylation, DNA alterations, N6-methyladenosine (m6a) RNA methylation, and cardiac mitochondrial mutations are explored as potential tools for precise stem cell differentiation.

More research is being conducted on myocardial cell treatments utilizing stem cell lines that can develop into cardiomyocytes. All of the forms of cardiac illnesses have shown to be quite amenable to treatments using embryonic (ESCs) and induced pluripotent stem cells (iPSCs). In the present study, we reviewed the differentiation of these cell types into cardiomyocytes from an epigenetic standpoint. We also provided a miRNA network that is devoted to the epigenetic commitment of stem cells toward cardiomyocyte cells and related diseases, such as congenital heart defects, comprehensively. Histone acetylation, methylation, DNA alterations, N6-methyladenosine (m6a) RNA methylation, and cardiac mitochondrial mutations are explored as potential tools for precise stem cell differentiation.

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90–1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82–0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

Growth factor signaling is a key determinant of cellular decisions ranging from stem cell fate, to metabolic behavior. As such, gaining control over these signaling events is of critical importance to the field of regenerative medicine, which is continuously seeking novel means to tailor the cellular microenvironment to direct cells towards medically advantageous outcomes. Heparan sulfate (HS) glycosaminoglycans (GAGS) are sulfated polysaccharides found on the cell surface and in the extracellular matrix which are responsible for the engagement of growth factors as well as growth factor receptors as a means to spatiotemporally direct cell signaling events.Despite the obvious potential associated with controlling HS GAG-growth factor interactions, HS GAG-based approaches to manipulate cellular signaling has been minimal due to the complex nature of this class of sulfated polysaccharides. Sulfated GAGs like HS have a non-template driven synthesis, that is, their structure is not dictated by a genetic blue print. During assembly, the GAGs undergo a series of sulfations, isomerizations, and acetylations which give rise to their specific binding capacity, but it is this same complexity that poses a significant hurdle for synthetic or chemoenzymatic approaches geared at producing these structures for study and medical application. As a result, novel approaches must be developed to hone control over cell signaling using GAGs while circumventing the structural obstacle posed by the complexity of their structure. In this dissertation, I present a variety of accessible methods for the preparation of synthetic HS GAG mimetics, and demonstrate their efficacy in several contexts. In chapter 2, I introduce a HS GAG presenting, membrane incorporating, polymer which utilizes a multivalent display of commercially available HS GAG disaccharides to exert control over stem cell fate in a structure dependent manner. In chapter 3 I reveal the role of HS GAG in mesodermal differentiation.

Growth factor signaling is a key determinant of cellular decisions ranging from stem cell fate, to metabolic behavior. As such, gaining control over these signaling events is of critical importance to the field of regenerative medicine, which is continuously seeking novel means to tailor the cellular microenvironment to direct cells towards medically advantageous outcomes. Heparan sulfate (HS) glycosaminoglycans (GAGS) are sulfated polysaccharides found on the cell surface and in the extracellular matrix which are responsible for the engagement of growth factors as well as growth factor receptors as a means to spatiotemporally direct cell signaling events.Despite the obvious potential associated with controlling HS GAG-growth factor interactions, HS GAG-based approaches to manipulate cellular signaling has been minimal due to the complex nature of this class of sulfated polysaccharides. Sulfated GAGs like HS have a non-template driven synthesis, that is, their structure is not dictated by a genetic blue print. During assembly, the GAGs undergo a series of sulfations, isomerizations, and acetylations which give rise to their specific binding capacity, but it is this same complexity that poses a significant hurdle for synthetic or chemoenzymatic approaches geared at producing these structures for study and medical application. As a result, novel approaches must be developed to hone control over cell signaling using GAGs while circumventing the structural obstacle posed by the complexity of their structure. In this dissertation, I present a variety of accessible methods for the preparation of synthetic HS GAG mimetics, and demonstrate their efficacy in several contexts. In chapter 2, I introduce a HS GAG presenting, membrane incorporating, polymer which utilizes a multivalent display of commercially available HS GAG disaccharides to exert control over stem cell fate in a structure dependent manner. In chapter 3 I reveal the role of HS GAG in mesodermal differentiation.

The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. Significance Statement Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems fo, QC 20221017

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

Survivin is one of the rare proteins that is differentially expressed in normal and cancer cells and is directly or indirectly involved in numerous pathways required for tumor maintenance. It is expressed in almost all cancers and its expression has been detected at early stages of cancer. These traits make survivin an exceptionally attractive target for cancer therapeutics. Even with these promising features to be an oncotherapeutic target, there has been limited success in the clinical trials targeting survivin. Only recently it has emerged that survivin was not being specifically targeted which could have resulted in the negative clinical outcome. Also, focus of research has now shifted from survivin expression in the overall heterogeneous tumor cell populations to survivin expression in cancer stem cells as these cells have proved to be the major drivers of tumors. Therefore, in this review we have analyzed the expression of survivin in normal and cancer cells with a particular focus on its expression in cancer stem cell compartment. We have discussed the major signaling pathways involved in regulation of survivin. We have explored the current development status of various types of interventions for inhibition of survivin. Furthermore, we have discussed the challenges involving the development of potent and specific survivin inhibitors for cancer therapeutics. Finally we have given insights for some of the promising future anticancer treatments., QC 20201214

Introduction: ETMRs are highly lethal, pediatric embryonal brain tumors, previously classified as various histologic diagnoses including supratentorial primitive neuroectodermal tumors (sPNET) and CNS PNET. With recognition that these tumors harbor recurrent amplification of a novel oncogenic miRNA cluster on chr19, C19MC, ETMRs were designated as a distinct biological and molecular entity with a spectrum of histologic and clinical manifestations. Method(s): We reviewed published literature describing clinical presentation, the genetic and epigenetic drivers of oncogenesis, and recent therapeutic strategies adopted to combat these aggressive tumors. Result(s): As a consequence of C19MC amplification, ETMRs upregulate several oncogenic and pluripotency proteins, including LIN28A, DNMT3B and MYCN, that confer a unique epigenetic signature reminiscent of nascent embryonic stem cells. In this review, we focus on the dysregulation of miRNAs in ETMR, the major pathogenic mechanism identified in this disease. Conclusion(s): Despite the use of multi-modal therapeutic regimens, ETMR patients have dismal survival. Understanding the unique biology of these tumors has provided new insights towards novel therapeutic targets.Copyright © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

Introduction: ETMRs are highly lethal, pediatric embryonal brain tumors, previously classified as various histologic diagnoses including supratentorial primitive neuroectodermal tumors (sPNET) and CNS PNET. With recognition that these tumors harbor recurrent amplification of a novel oncogenic miRNA cluster on chr19, C19MC, ETMRs were designated as a distinct biological and molecular entity with a spectrum of histologic and clinical manifestations. Method(s): We reviewed published literature describing clinical presentation, the genetic and epigenetic drivers of oncogenesis, and recent therapeutic strategies adopted to combat these aggressive tumors. Result(s): As a consequence of C19MC amplification, ETMRs upregulate several oncogenic and pluripotency proteins, including LIN28A, DNMT3B and MYCN, that confer a unique epigenetic signature reminiscent of nascent embryonic stem cells. In this review, we focus on the dysregulation of miRNAs in ETMR, the major pathogenic mechanism identified in this disease. Conclusion(s): Despite the use of multi-modal therapeutic regimens, ETMR patients have dismal survival. Understanding the unique biology of these tumors has provided new insights towards novel therapeutic targets.Copyright © 2020, Springer Science+Business Media, LLC, part of Springer Nature.

Amyotrophic lateral sclerosis (ALS) is a devastating incurable neurological disorder characterized by motor neuron (MN) death and muscle dysfunction leading to mean survival time after diagnosis of only 2–5 years. A potential ALS treatment is to delay the loss of MNs and disease progression by the delivery of trophic factors. Previously, we demonstrated that implanted mesoporous silica nanoparticles (MSPs) loaded with trophic factor peptide mimetics support survival and induce differentiation of co-implanted embryonic stem cell (ESC)-derived MNs. Here, we investigate whether MSP loaded with peptide mimetics of ciliary neurotrophic factor (Cintrofin), glial-derived neurotrophic factor (Gliafin), and vascular endothelial growth factor (Vefin1) injected into the cervical spinal cord of mutant SOD1 mice affect disease progression and extend survival. We also transplanted boundary cap neural crest stem cells (bNCSCs) which have been shown previously to have a positive effect on MN survival in vitro and in vivo. We show that mimetic-loaded MSPs and bNCSCs significantly delay disease progression and increase survival of mutant SOD1 mice, and also that empty particles significantly improve the condition of ALS mice. Our results suggest that intraspinal delivery of MSPs is a potential therapeutic approach for the treatment of ALS., QC 20211002

The availability of appropriate and reliable in vitro cell models recapitulating human cardiovascular diseases has been the aim of numerous researchers, in order to retrace pathologic phenotypes, elucidate molecular mechanisms, and discover therapies using simple and reproducible techniques. In the past years, several human cell types have been utilized for these goals, including heterologous systems, cardiovascular and non-cardiovascular primary cells, and embryonic stem cells. The introduction of induced pluripotent stem cells and their differentiation potential brought new prospects for large-scale cardiovascular experiments, bypassing ethical concerns of embryonic stem cells and providing an advanced tool for disease modeling, diagnosis, and therapy. Each model has its advantages and disadvantages in terms of accessibility, maintenance, throughput, physiological relevance, recapitulation of the disease. A higher level of complexity in diseases modeling has been achieved with multicellular co-cultures. Furthermore, the important progresses reached by bioengineering during the last years, together with the opportunities given by pluripotent stem cells, have allowed the generation of increasingly advanced in vitro three-dimensional tissue-like constructs mimicking in vivo physiology. This review provides an overview of the main cell models used in cardiovascular research, highlighting the pros and cons of each, and describing examples of practical applications in disease modeling.

Atypical teratoid/rhabdoid tumor (AT/RT), which harbors SMARCB1 mutation and exhibits a characteristic histology of rhabdoid cells, has a poor prognosis because of the lack of effective treatments. Here, we establish human SMARCB1-deficient pluripotent stem cells (hPSCs). SMARCB1-deficient hPSC-derived neural progenitor-like cells (NPLCs) efficiently give rise to brain tumors when transplanted into the mouse brain. Notably, activation of an embryonic stem cell (ESC)-like signature confers a rhabdoid histology in SMARCB1-deficient NPLC-derived tumors and causes a poor prognosis. Consistently, we find the activation of the ESC-like gene expression signature and an ESC-like DNA methylation landscape in clinical specimens of AT/RT. Finally, we identify candidate genes that maintain the activation of the ESC-like signature and the growth of AT/RT cells. Collectively, SMARCB1-deficient hPSCs offer the human models for AT/RT, which uncover the role of the activated ESC-like signature in the poor prognosis and unique histology of AT/RT.

Chemical modifications of RNA provide a direct and rapid way to modulate the existing transcriptome, allowing the cells to adapt rapidly to the changing environment. Among these modifications, N6-methyladenosine (m6A) has recently emerged as a widely prevalent mark of messenger RNA in eukaryotes, linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. m6A modification modulates a broad spectrum of biochemical processes, including mRNA decay, translation and splicing. Both m6A modification and the enzymes that control m6A metabolism are essential for normal development. In this review, we summarized the most recent findings on the role of m6A modification in maintenance of the pluripotency of embryonic stem cells (ESCs), cell fate specification, the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), and differentiation of stem and progenitor cells., This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert

The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by spatially and temporally varying morphogen signals. Such dynamic morphogen patterns drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. In embryonic stem cell (ESC) models for early development, for example, analogous tissue patterns spontaneously emerge but mechanistic insight into this self-organization has been limited. In this dissertation, we combine engineered methods for optogenetic stimulation and biological analysis to photoactivate Wnt/B-catenin signaling and to study morphogen dynamics and self-organization of human pluripotent stem cells. First, we show that optogenetic Wnt/B-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns in human ESCs (hESCs). We expressed a fusion of the plant blue-light photoreceptor Cryptochrome 2 (Cry2) to the Wnt co-receptor LRP6 (the optoWnt system) in hESCs and induce Cry2-LRP6c oligomerization and subsequent canonical Wnt/B-catenin activation using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). Such LAVA devices are a LED-based, programmable illumination system for photostimulation of multi-well plates that can be readily incorporated into the workflow of routine cell culture and allow controlled and quantitative spatiotemporal light patterning. We optimized the LAVA board optical configuration for illumination uniformity and achieve programmable photostimulation of independent wells of 24-well or 96-well culture plates kept in standard 37C tissue culture incubators. Each well can be wirelessly programmed through a graphical user in

Human natural killer (NK) cell-based adoptive anticancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if needed. These characteristics make hESC-/iPSC-derived NK cells an ideal cell population for developing standardized, "off-the-shelf" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously has established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin (IL)-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single-cell adaptation of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESCs/hiPSCs are directly used to form embryoid body (EB) to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by spatially and temporally varying morphogen signals. Such dynamic morphogen patterns drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. In embryonic stem cell (ESC) models for early development, for example, analogous tissue patterns spontaneously emerge but mechanistic insight into this self-organization has been limited. In this dissertation, we combine engineered methods for optogenetic stimulation and biological analysis to photoactivate Wnt/B-catenin signaling and to study morphogen dynamics and self-organization of human pluripotent stem cells. First, we show that optogenetic Wnt/B-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns in human ESCs (hESCs). We expressed a fusion of the plant blue-light photoreceptor Cryptochrome 2 (Cry2) to the Wnt co-receptor LRP6 (the optoWnt system) in hESCs and induce Cry2-LRP6c oligomerization and subsequent canonical Wnt/B-catenin activation using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). Such LAVA devices are a LED-based, programmable illumination system for photostimulation of multi-well plates that can be readily incorporated into the workflow of routine cell culture and allow controlled and quantitative spatiotemporal light patterning. We optimized the LAVA board optical configuration for illumination uniformity and achieve programmable photostimulation of independent wells of 24-well or 96-well culture plates kept in standard 37C tissue culture incubators. Each well can be wirelessly programmed through a graphical user in

Due to their potential for tissue engineering applications and ability to modulate the immune system and reduce inflammation, mesenchymal stem cells (MSCs) have been explored as a promising option for the treatment of chronic diseases and injuries. However, there are problems associated with the use of this type of cell that limit their applications. Several studies have been exploring the possibility to produce mesenchymal stem cells from pluripotent stem cells (PSCs). The aim of these studies is to generate MSCs with advantageous characteristics of both PSCs and MSCs. However, there are still some questions concerning the characteristics of MSCs derived from the differentiation of PSCs that must be answered before they can be used to treat diseases and injuries. The objective of this study was, therefore, to determine if PSCs exposed to SB431542, a TGF-beta inhibitor, are able to differentiate to MSCs, judging by morphology, expression of mesenchymal and pluripotent stem cell markers, expression of pluripotency-related genes, and ability to differentiate to osteocytes and adipocytes. The results obtained demonstrated that it is possible to induce the differentiation of both embryonic stem cells and induce pluripotent stem cells into cells with characteristics that highly resemble those from MSCs through the inhibition of the TGF-beta pathway.Copyright © 2018 Alessander Leyendecker Junior.

Due to their potential for tissue engineering applications and ability to modulate the immune system and reduce inflammation, mesenchymal stem cells (MSCs) have been explored as a promising option for the treatment of chronic diseases and injuries. However, there are problems associated with the use of this type of cell that limit their applications. Several studies have been exploring the possibility to produce mesenchymal stem cells from pluripotent stem cells (PSCs). The aim of these studies is to generate MSCs with advantageous characteristics of both PSCs and MSCs. However, there are still some questions concerning the characteristics of MSCs derived from the differentiation of PSCs that must be answered before they can be used to treat diseases and injuries. The objective of this study was, therefore, to determine if PSCs exposed to SB431542, a TGF-beta inhibitor, are able to differentiate to MSCs, judging by morphology, expression of mesenchymal and pluripotent stem cell markers, expression of pluripotency-related genes, and ability to differentiate to osteocytes and adipocytes. The results obtained demonstrated that it is possible to induce the differentiation of both embryonic stem cells and induce pluripotent stem cells into cells with characteristics that highly resemble those from MSCs through the inhibition of the TGF-beta pathway.Copyright © 2018 Alessander Leyendecker Junior.

The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO and occurs in approximately 10% of all acute myeloid leukaemias. RUNX1-ETO transcripts can be detected in utero and in cells of patients in remission, but its sole expression is insufficient to cause overt leukaemia. Given that t(8;21) patient cells present additional mutations, the epigenetic reprogramming directly mediated by RUNX1-ETO remains unclear. To address this question, we generated human Embryonic Stem Cell lines carrying an inducible RUNX1-ETO transgene, which we subsequently differentiated into definitive haematopoietic progenitors. We show that induction of RUNX1-ETO in already formed progenitors (i) blocks differentiation at an immature stage, (ii) induces a cell-type specific and reversible cell cycle arrest, (iii) abrogates the RUNX1-mediated gene expression program by interfering with RUNX1 binding, resulting in downregulation of haematopoietic, cell cycle as well as DNA repair genes, (iv) closes down a large part of the chromatin accessibility pattern present in adult haematopoietic multipotent progenitors and (v) alters the differentiation of a defined sub-population of progenitors. Our data are consistent with the idea that RUNX1-ETO establishes a precondition for leukaemic transformation by maintaining a reservoir of quiescent pre-leukaemic multipotent progenitors with susceptibility to expand upon acquisition of additional oncogenic events.

The processes of cell proliferation, differentiation, migration, and self-organization during early embryonic development are governed by spatially and temporally varying morphogen signals. Such dynamic morphogen patterns drive cell fate specification at the proper location and time. However, current in vitro methods typically do not allow for precise, dynamic, spatiotemporal control of morphogen signaling and are thus insufficient to readily study how morphogen dynamics impact cell behavior. In embryonic stem cell (ESC) models for early development, for example, analogous tissue patterns spontaneously emerge but mechanistic insight into this self-organization has been limited. In this dissertation, we combine engineered methods for optogenetic stimulation and biological analysis to photoactivate Wnt/B-catenin signaling and to study morphogen dynamics and self-organization of human pluripotent stem cells. First, we show that optogenetic Wnt/B-catenin pathway activation can be controlled at user-defined intensities, temporal sequences, and spatial patterns in human ESCs (hESCs). We expressed a fusion of the plant blue-light photoreceptor Cryptochrome 2 (Cry2) to the Wnt co-receptor LRP6 (the optoWnt system) in hESCs and induce Cry2-LRP6c oligomerization and subsequent canonical Wnt/B-catenin activation using novel engineered illumination devices for optogenetic photostimulation and light activation at variable amplitudes (LAVA). Such LAVA devices are a LED-based, programmable illumination system for photostimulation of multi-well plates that can be readily incorporated into the workflow of routine cell culture and allow controlled and quantitative spatiotemporal light patterning. We optimized the LAVA board optical configuration for illumination uniformity and achieve programmable photostimulation of independent wells of 24-well or 96-well culture plates kept in standard 37C tissue culture incubators. Each well can be wirelessly programmed through a graphical user in

Human natural killer (NK) cell-based adoptive anticancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if needed. These characteristics make hESC-/iPSC-derived NK cells an ideal cell population for developing standardized, "off-the-shelf" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously has established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin (IL)-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single-cell adaptation of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESCs/hiPSCs are directly used to form embryoid body (EB) to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

Somites are periodically formed by segmentation of the anterior parts of the presomitic mesoderm (PSM). In the mouse embryo, this periodicity is controlled by the segmentation clock gene Hes7, which exhibits wave-like oscillatory expression in the PSM. Despite intensive studies, the exact mechanism of such synchronous oscillatory dynamics of Hes7 expression still remains to be analyzed. Detailed analysis of the segmentation clock has been hampered because it requires the use of live embryos, and establishment of an in vitro culture system would facilitate such analyses. Here, we established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in which Hes7 expression oscillates like traveling waves. In these tissues, Hes7 oscillation is synchronized between neighboring cells, and the posterior-anterior axis is self-organized as the central-peripheral axis. This method is applicable to chemical-library screening and will facilitate the analysis of the molecular nature of the segmentation clock.

Serine/threonine kinase 35 (STK35) is a recently identified human kinase with an autophosphorylation function, linked functionally to actin stress fibers, cell cycle progression and survival. STK35 has previously been shown to be highly expressed in human testis, and we demonstrated its regulation by nuclear-localized importin alpha2 in HeLa cells. The present study identifies progressive expression from the STK35 locus of two coding mRNA isoforms and one long noncoding RNA (lncRNA) in mouse testis during spermatogenesis, indicating their tightly controlled synthesis. Additionally, lncRNA transcripts are increased by exposure to oxidative stress in mouse GC-1 germ cell line. STK35 knockout (KO) mice lacking all three RNAs are born at sub-Mendelian frequency, and adults manifest both male and female germline deficiency. KO males exhibit no or partial spermatogenesis in most testis tubule cross-sections; KO ovaries are smaller and contain fewer follicles. Eyes of KO mice display phenotypes ranging from gross deformity to mild goniodysgenesis or iridocorneal angle malformation, to overtly normal. These findings demonstrate the tight regulation of transcription from the STK35 locus and its central importance to fertility, eye development and cell responses to oxidative stress.Copyright © 2018. Published by The Company of Biologists Ltd.

Serine/threonine kinase 35 (STK35) is a recently identified human kinase with an autophosphorylation function, linked functionally to actin stress fibers, cell cycle progression and survival. STK35 has previously been shown to be highly expressed in human testis, and we demonstrated its regulation by nuclear-localized importin alpha2 in HeLa cells. The present study identifies progressive expression from the STK35 locus of two coding mRNA isoforms and one long noncoding RNA (lncRNA) in mouse testis during spermatogenesis, indicating their tightly controlled synthesis. Additionally, lncRNA transcripts are increased by exposure to oxidative stress in mouse GC-1 germ cell line. STK35 knockout (KO) mice lacking all three RNAs are born at sub-Mendelian frequency, and adults manifest both male and female germline deficiency. KO males exhibit no or partial spermatogenesis in most testis tubule cross-sections; KO ovaries are smaller and contain fewer follicles. Eyes of KO mice display phenotypes ranging from gross deformity to mild goniodysgenesis or iridocorneal angle malformation, to overtly normal. These findings demonstrate the tight regulation of transcription from the STK35 locus and its central importance to fertility, eye development and cell responses to oxidative stress.Copyright © 2018. Published by The Company of Biologists Ltd.

Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions.

The rate of glycolytic metabolism changes during differentiation of human embryonic stem cells (hESCs) and reprogramming of somatic cells to pluripotent stem cells. However, the functional contribution of glycolytic metabolism to pluripotency is unclear. Here we show that the degree of pluripotency is associated with glycolytic rate, whereby naive hESCs exhibit increased glycolytic flux, MYC transcriptional activity, and nuclear localization of N-MYC relative to primed hESCs. This is consistent with the inner cell mass of human blastocysts which exhibit increased MYC transcriptional activity relative primed hESCs and elevated nuclear N-MYC levels. Reduction of glycolysis decreases self-renewal of naive hESCs and feeder-free cultured primed hESCs, but not primed hESCs grown in feeder-supported conditions. Reduction of glycolysis in feeder-free primed hESCs also enhances neural specification. These findings reveal associations between glycolytic metabolism and the state of pluripotency, differences in the metabolism of feeder- versus feeder-free cultured hESCs, and identify methods for regulating self-renewal and initial cell fate specification of hESCs.

The rate of glycolytic metabolism changes during differentiation of human embryonic stem cells (hESCs) and reprogramming of somatic cells to pluripotent stem cells. However, the functional contribution of glycolytic metabolism to pluripotency is unclear. Here we show that the degree of pluripotency is associated with glycolytic rate, whereby naive hESCs exhibit increased glycolytic flux, MYC transcriptional activity, and nuclear localization of N-MYC relative to primed hESCs. This is consistent with the inner cell mass of human blastocysts which exhibit increased MYC transcriptional activity relative primed hESCs and elevated nuclear N-MYC levels. Reduction of glycolysis decreases self-renewal of naive hESCs and feeder-free cultured primed hESCs, but not primed hESCs grown in feeder-supported conditions. Reduction of glycolysis in feeder-free primed hESCs also enhances neural specification. These findings reveal associations between glycolytic metabolism and the state of pluripotency, differences in the metabolism of feeder- versus feeder-free cultured hESCs, and identify methods for regulating self-renewal and initial cell fate specification of hESCs.

The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions.

Background & Objective: Despite the great effort spent over recent decades to unravel the pathological mechanisms underpinning the development of central nervous system disorders, most of them still remain unclear. In particular, the study of rare CNS diseases is hampered by the lack of post-mortem samples and of reliable epidemiological studies, thus the setting of in vitro modeling systems appears essential to dissect the puzzle of genetic and environmental alterations affecting neural cells viability and functionality. The isolation and expansion in vitro of embryonic (ESC) and fetal neural stem cells (NSC) from human tissue have allowed the modeling of several neurological diseases “in a dish” and have also provided a novel platform to test potential therapeutic strategies in a pre-clinical setting. In recent years, the development of induced pluripotent stem cell (iPS) technology has added enormous value to the aforementioned approach, thanks to their capability for generating disease-relevant cell phenotypes in vitro and to their perspective use in autologous transplantation. However, while the potentiality of ESC, NSC and iPS has been widely sponsored, the pitfalls related to the available protocols for differentiation and the heterogeneity of lines deriving from different individuals have been poorly discussed. Here we present pro and contra of using ESC, NSC or iPS for modeling rare diseases like Lysosomal Storage disorders and Motor Neuron Diseases. Conclusion: In this view, the advent of gene editing technologies is a unique opportunity to standardize the data analysis in preclinical studies and to tailor clinical protocols for stem cell-mediated therapy.

Both embryonic and adult stem cells are endowed with a superior capacity to prevent the accumulation of genetic lesions, repair them, or avoid their propagation to daughter cells, which would be particularly detrimental to the whole organism. Inducible pluripotent stem cells also display a robust DNA damage response, but the stability of their genome is often conditioned by the mutational history of the cell population of origin, which constitutes an obstacle to clinical applications. Cancer stem cells are particularly tolerant to DNA damage and fail to undergo senescence or regulated cell death upon accumulation of genetic lesions. Such a resistance contributes to the genetic drift of evolving tumors as well as to their limited sensitivity to chemo- and radiotherapy. Here, we discuss the pathophysiological and therapeutic implications of the molecular pathways through which stem cells cope with DNA damage.

Three-dimensional organoid constructs serve as increasingly widespread in vitro models for development and disease modeling. Current approaches to recreate morphogenetic processes in vitro rely on poorly controllable and ill-defined matrices, thereby largely overlooking the contribution of biochemical and biophysical extracellular matrix (ECM) factors in promoting multicellular growth and reorganization. Here, we show how defined synthetic matrices can be used to explore the role of the ECM in the development of complex 3D neuroepithelial cysts that recapitulate key steps in early neurogenesis. We demonstrate how key ECM parameters are involved in specifying cytoskeleton-mediated symmetry-breaking events that ultimately lead to neural tube-like patterning along the dorsal-ventral (DV) axis. Such synthetic materials serve as valuable tools for studying the discrete action of extrinsic factors in organogenesis, and allow for the discovery of relationships between cytoskeletal mechanobiology and morphogenesis.

Glycosaminoglycan (GAG) mediated growth factor signaling initiates differentiation in embryonic stem cells (ESCs). Manipulation of the GAG interactome affords a novel tool to understand and direct stem cell differentiation. In this study, a high-throughput miniaturized cellular microarray platform, consisting of gelatin robotically spotted onto passivated glass slides is established to assess the effects of cellular glycan microenviroment on growth factor signaling in mouse embryonic stem cells (mESCs). Heparan sulfate (HS) acts as co-receptor for fibroblast growth factor 2 (FGF2) and the subsequent activation of the mitogen activated protein kinase (MAPK) pathway is measured by extracellular regulated kinase (ERK) phosphorylation. Using HS deficient EXT1-/-¬ ESCs, FGF2 signaling is rescued using soluble and immobilized heparin. Immobilization of glycans on cellular microarrays offer potential as a powerful tool for systematically evaluating and directing cellular differentiation.Then, using conventional culture conditions, the interactions of HS and FGF2 are screened using bis-2-methyl-4-amino-quinolyl-6-carbamide, or surfen, and compared to the commercial FGFR inhibitor PD173074. Surfen reversibly antagonizes heparan sulfate in the µm range and can HS mediated interactions can be rescued using soluble heparin, a highly sulfated HS analog.

Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.Copyright © 2015 AJA, SIMM & SJTU. All rights reserved.

Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.Copyright © 2015 AJA, SIMM & SJTU. All rights reserved.

Glycosaminoglycan (GAG) mediated growth factor signaling initiates differentiation in embryonic stem cells (ESCs). Manipulation of the GAG interactome affords a novel tool to understand and direct stem cell differentiation. In this study, a high-throughput miniaturized cellular microarray platform, consisting of gelatin robotically spotted onto passivated glass slides is established to assess the effects of cellular glycan microenviroment on growth factor signaling in mouse embryonic stem cells (mESCs). Heparan sulfate (HS) acts as co-receptor for fibroblast growth factor 2 (FGF2) and the subsequent activation of the mitogen activated protein kinase (MAPK) pathway is measured by extracellular regulated kinase (ERK) phosphorylation. Using HS deficient EXT1-/-¬ ESCs, FGF2 signaling is rescued using soluble and immobilized heparin. Immobilization of glycans on cellular microarrays offer potential as a powerful tool for systematically evaluating and directing cellular differentiation.Then, using conventional culture conditions, the interactions of HS and FGF2 are screened using bis-2-methyl-4-amino-quinolyl-6-carbamide, or surfen, and compared to the commercial FGFR inhibitor PD173074. Surfen reversibly antagonizes heparan sulfate in the µm range and can HS mediated interactions can be rescued using soluble heparin, a highly sulfated HS analog.

The establishment of permanent human embryonic stem cell lines (hESCs) was first reported in 1998. Due to their pluripotent nature and ability to differentiate to all cell types in the body, they have been considered as a cell source for regenerative medicine. Since then, intensive studies have been carried out regarding factors regulating pluripotency and differentiation. hESCs are obtained from supernumerary human IVF (in vitro fertilization) embryos that cannot be used for the couple's infertility treatment. Today, we can establish and expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos. There are satisfactory tests for the demonstration of genetic stability, absence of tumorigenic mutations, functionality, and safety of hESCs. Clinical trials are ongoing for age-related macular degeneration (AMD) and spinal cord injury (SCI). This review focuses on the present state of these techniques.

Degenerative conditions of the lumbar spine are extremely common. Ninety percent of people over the age of 60 years have degenerative change on imaging; however, only a small minority of people will require spine surgery (Hicks et al. Spine (Phila Pa 1976) 34(12):1301-1306, 2009). This minority, however, constitutes a core element of spinal surgery practice. Whilst the patient outcomes from spinal surgeries have improved in recent years, some patients will remain with pain and disability despite technically successful surgery. Advances in regenerative medicine and stem cell therapies, particularly the use of mesenchymal stem cells and allogeneic mesenchymal precursor cells, have led to numerous clinical trials utilising these cell-based therapies to treat degenerative spinal conditions. Through cartilage formation and disc regeneration, fusion enhancement or via modification of pain pathways, stem cells are well suited to enhance spinal surgery practice. This review will focus on the outcomes of lumbar spinal procedures and the role of stem cells in the treatment of degenerative lumbar conditions to enhance clinical practice. The current status of clinical trials utilising stem cell therapies will be discussed, providing clinicians with an overview of the various cell-based treatments likely to be available to patients in the near future.Copyright © 2015, Springer-Verlag Berlin Heidelberg.

Degenerative conditions of the lumbar spine are extremely common. Ninety percent of people over the age of 60 years have degenerative change on imaging; however, only a small minority of people will require spine surgery (Hicks et al. Spine (Phila Pa 1976) 34(12):1301-1306, 2009). This minority, however, constitutes a core element of spinal surgery practice. Whilst the patient outcomes from spinal surgeries have improved in recent years, some patients will remain with pain and disability despite technically successful surgery. Advances in regenerative medicine and stem cell therapies, particularly the use of mesenchymal stem cells and allogeneic mesenchymal precursor cells, have led to numerous clinical trials utilising these cell-based therapies to treat degenerative spinal conditions. Through cartilage formation and disc regeneration, fusion enhancement or via modification of pain pathways, stem cells are well suited to enhance spinal surgery practice. This review will focus on the outcomes of lumbar spinal procedures and the role of stem cells in the treatment of degenerative lumbar conditions to enhance clinical practice. The current status of clinical trials utilising stem cell therapies will be discussed, providing clinicians with an overview of the various cell-based treatments likely to be available to patients in the near future.Copyright © 2015, Springer-Verlag Berlin Heidelberg.

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy, disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers, urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen, Chlorpromazine, Diclofenac, Digoxin, Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore, SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development.

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell-cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here, we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions, resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions, which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity.

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time, cost and high inter-species variability. Here, we developed a human-relevant in vitro model, which recapitulated two cellular events characteristic of embryogenesis, to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation, epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human.

Our ancestors and our children are linked by a single, special cell lineage called the germline. Germ cells are tasked with the role to accurately pass DNA from one generation to the next. In today's society infertility is an important health concern as it is estimated to affect 10% of the reproductive age population. In many cases infertility can be traced to abnormal germline cell development. The early events of germline formation are difficult to study because of limited materials, especially low germ cell numbers in the process of embryogenesis.To overcome the challenge of limited materials, an in vitro method to derive cell types faithfully reporting in vivo phenotypes needs to be devised to facilitate the research process. The key results from the in vitro model should then be evaluated carefully in vivo to show relevance in development. My thesis first focused on establishing an in vitro model to recapitulate early development of germline, by differentiating embryonic stem cells (ESCs) to form in vitro primordial germ cells (iPGCs), in a dish. This method allows good number of germ cells to be produced for molecular and biochemical studies. Using the in vitro model, key germline modifiers for ESC maintenance and germ cell formation were identified, one of which is protein arginine methyltransferase 5 (PRMT5). PRMT5 is a type II arginine methyltransferase (PRMT) that modifies symmetrical dimethylated arginines (SDMAs) on proteins, which substrates include histone H2A and H4, as well as the Sm proteins involved in RNA splicing. However, the molecular function of PRMT5 in ground state ESCs remains unknown. In our study, for the first time, we generated an inducible knock out of Prmt5 in ESCs cultured in ground state pluripotency (cultured with 2i inhibitors and leukemia inhibitory factor). We characterized PRMT5's role in ESCs through the method of paired-end RNA sequencing to show that PRMT5 affects cell survival, proliferation and chromatin organization etc.

Pluripotency establishment and maintenance are critical for both the development of mammalian embryos and the production of pluripotent stem cells. Investigations into the molecular mechanisms underlying pluripotency have revealed multiple signaling networks that generate diverse pluripotent states. The relationship between these networks, their upstream transcription factors, and their associated pluripotent states is complex and poorly understood in both pluripotent stem cells and embryos. In this thesis I examine three aspects of pluripotency regulation: the role of maternal transcription factors in embryogenesis, novel effects of genetic background and naivety on differentiation to placental stem cells, and the contribution of known stem cell factors to regulation of pluripotency in vivo.

The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self-renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c-Myc, Oct4, Sox2, and Klf4) to form embryonic stem cell-like cells (ESCLCs), which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate toward epiblast-like cells (EpiLCs) and primordial germ cell-like cells (PGCLCs) by culturing in media supplied with activin A and BMP-4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2-3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop-like colonies, and the expression of pluripotency-related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4-6 days, they formed typical water drop-like colonies again. Moreover, expression of the pluripotency-related genes and the primordial germ cell (PGC) specification-related genes increased. During progression from ESCLCs toward EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in PGC specification. The derived PGCLCs expressed SSEA-1, Blimp-1, and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non-PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure. © 2014 International Federation for Cell Biology.