Stem Cells and Infertility: A Review of Clinical Applications and Legal Frameworks.

Infertility is a condition defined by the failure to establish a clinical pregnancy after 12 months of regular, unprotected sexual intercourse or due to an impairment of a person's capacity to reproduce either as an individual or with their partner. The authors have set out to succinctly investigate, explore, and assess infertility treatments, harnessing the potential of stem cells to effectively and safely treat infertility; in addition, this paper will present the legal and regulatory complexities at the heart of stem cell research, with an overview of the legislative state of affairs in six major European countries. For couples who cannot benefit from assisted reproductive technologies (ART) to treat their infertility, stem-cells-based approaches have been shown to be a highly promising approach. Nonetheless, lingering ethical and immunological uncertainties require more conclusive findings and data before such treatment avenues can become mainstream and be applied on a large scale. The isolation of human embryonic stem cells (ESCs) is ethically controversial, since their collection involves the destruction of human embryonic tissue. Overall, stem cell research has resulted in important new breakthroughs in the treatment of infertility. The effort to untangle the complex web of ethical and legal issues associated with such therapeutic approaches will have to rely on evidence-based, broadly shared standards, guidelines, and best practices to make sure that the procreative rights of patients can be effectively reconciled with the core values at the heart of medical ethics.

Protective role of stem cells in POI: Current status and mechanism of action, a review article.

Premature ovarian insufficiency (POI) has far-reaching consequences on women's life quality. Due to the lack of full recognition of the etiology and complexity of this disease, there is no appropriate treatment for infected patients. Recently, stem cell therapy has attracted the attention of regenerative medicine scholars and offered promising outcomes for POI patients. Several kinds of stem cells, such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and induced pluripotent stem cells (iPSCs) have been used for the treatment of ovarian diseases. However, their potential protective mechanisms are still unknown. Undoubtedly, a better understanding of the therapeutic molecular and cellular mechanisms of stem cells will address uncover strategies to increase their clinical application for multiple disorders such as POI. This paper describes a detailed account of the potential properties of different types of stem cells and provides a comprehensive review of their protective mechanisms, particularly MSC, in POI disorder. In addition, ongoing challenges and several strategies to improve the efficacy of MSC in clinical use are addressed. Therefore, this review will provide proof-of-concept for further clinical application of stem cells in POI.

RNA polymerase II transcription with partially assembled TFIID complexes.

The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.

Regulatory Elements Outside Established Pou5f1 Gene Boundaries Are Required for Multilineage Differentiation of Embryonic Stem Cells.

The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.

Metabolic and cell cycle shift induced by the deletion of Dnm1l attenuates the dissolution of pluripotency in mouse embryonic stem cells.

Mitochondria are versatile organelles that continuously change their morphology via fission and fusion. However, the detailed functions of mitochondrial dynamics-related genes in pluripotent stem cells remain largely unclear. Here, we aimed to determine the effects on energy metabolism and differentiation ability of mouse embryonic stem cells (ESCs) following deletion of the mitochondrial fission-related gene Dnml1. Resultant Dnm1l-/- ESCs maintained major pluripotency characteristics. However, Dnm1l-/- ESCs showed several phenotypic changes, including the inhibition of differentiation ability (dissolution of pluripotency). Notably, Dnm1l-/- ESCs maintained the expression of the pluripotency marker Oct4 and undifferentiated colony types upon differentiation induction. RNA sequencing analysis revealed that the most frequently differentially expressed genes were enriched in the glutathione metabolic pathway. Our data suggested that differentiation inhibition of Dnm1l-/- ESCs was primarily due to metabolic shift from glycolysis to OXPHOS, G2/M phase retardation, and high level of Nanog and 2-cell-specific gene expression.

Identification and characterization of enhancer elements controlling cell type-specific and signalling dependent chromatin programming during hematopoietic development.

The development of multi-cellular organisms from a single fertilized egg requires to differentially execute the information encoded in our DNA. This complex process is regulated by the interplay of transcription factors with a chromatin environment, both of which provide the epigenetic information maintaining cell-type specific gene expression patterns. Moreover, transcription factors and their target genes form vast interacting gene regulatory networks which can be exquisitely stable. However, all developmental processes originate from pluripotent precursor cell types. The production of terminally differentiated cells from such cells, therefore, requires successive changes of cell fates, meaning that genes relevant for the next stage of differentiation must be switched on and genes not relevant anymore must be switched off. The stimulus for the change of cell fate originates from extrinsic signals which set a cascade of intracellular processes in motion that eventually terminate at the genome leading to changes in gene expression and the development of alternate gene regulatory networks. How developmental trajectories are encoded in the genome and how the interplay between intrinsic and extrinsic processes regulates development is one of the major questions in developmental biology. The development of the hematopoietic system has long served as model to understand how changes in gene regulatory networks drive the differentiation of the various blood cell types. In this review, we highlight the main signals and transcription factors and how they are integrated at the level of chromatin programming and gene expression control. We also highlight recent studies identifying the cis-regulatory elements such as enhancers at the global level and explain how their developmental activity is regulated by the cooperation of cell-type specific and ubiquitous transcription factors with extrinsic signals.

Generation of Oligodendrocytes from Human Pluripotent and Embryonic Stem Cells.

Oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes (OLs) can be generated using human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). By manipulating culture conditions, pluripotent cell types are serially guided through intermediary cell types, developing first into neural progenitor cells (NPCs) then OPCs before maturing as CNS-specific OLs. This procedure is conducted under adherent, feeder-free conditions to derive mature OLs in as few as 28 days.

Single-Cell and Spatial Analysis of Emergent Organoid Platforms.

Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

The regulation of transcription elongation in embryonic stem cells.

Transcription elongation is a fundamental molecular process which is accurately regulated to ensure proper gene expression in cellular activities whereas its malfunction is associated with impaired cellular functions. Embryonic stem cells (ESCs) have significant value in regenerative medicine due to their self-renewal ability and their potential to differentiate to almost all types of cells. Therefore, dissection of the exact regulatory mechanism of transcription elongation in ESCs is crucial for both basic research and their clinical applications. In this review, we discuss the current understanding on the regulatory mechanisms of transcription elongation mediated by transcription factors and epigenetic modifications in ESCs.

Metabolic control of induced pluripotency.

Pluripotent stem cells of the mammalian epiblast and their cultured counterparts-embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs)-have the capacity to differentiate in all cell types of adult organisms. An artificial process of reactivation of the pluripotency program in terminally differentiated cells was established in 2006, which allowed for the generation of induced pluripotent stem cells (iPSCs). This iPSC technology has become an invaluable tool in investigating the molecular mechanisms of human diseases and therapeutic drug development, and it also holds tremendous promise for iPSC applications in regenerative medicine. Since the process of induced reprogramming of differentiated cells to a pluripotent state was discovered, many questions about the molecular mechanisms involved in this process have been clarified. Studies conducted over the past 2 decades have established that metabolic pathways and retrograde mitochondrial signals are involved in the regulation of various aspects of stem cell biology, including differentiation, pluripotency acquisition, and maintenance. During the reprogramming process, cells undergo major transformations, progressing through three distinct stages that are regulated by different signaling pathways, transcription factor networks, and inputs from metabolic pathways. Among the main metabolic features of this process, representing a switch from the dominance of oxidative phosphorylation to aerobic glycolysis and anabolic processes, are many critical stage-specific metabolic signals that control the path of differentiated cells toward a pluripotent state. In this review, we discuss the achievements in the current understanding of the molecular mechanisms of processes controlled by metabolic pathways, and vice versa, during the reprogramming process.

Using Pluripotent Stem Cells to Understand Normal and Leukemic Hematopoietic Development.

Several decades have passed since the generation of the first embryonic stem cell (ESC) lines both in mice and in humans. Since then, stem cell biologists have tried to understand their potential biological and clinical uses for their implementation in regenerative medicine. The hematopoietic field was a pioneer in establishing the potential use for the development of blood cell products and clinical applications; however, early expectations have been truncated by the difficulty in generating bonafide hematopoietic stem cells (HSCs). Despite some progress in understanding the origin of HSCs during embryonic development, the reproduction of this process in vitro is still not possible, but the knowledge acquired in the embryo is slowly being implemented for mouse and human pluripotent stem cells (PSCs). In contrast, ESC-derived hematopoietic cells may recapitulate some leukemic transformation processes when exposed to oncogenic drivers. This would be especially useful to model prenatal leukemia development or other leukemia-predisposing syndromes, which are difficult to study. In this review, we will review the state of the art of the use of PSCs as a model for hematopoietic and leukemia development.

Long Noncoding RNA Lx8-SINE B2 Interacts with Eno1 to Regulate Self-Renewal and Metabolism of Embryonic Stem Cells.

Long noncoding RNAs (lncRNAs) emerge as important orchestrators of biological processes in embryonic stem cells (ESCs). LncRNA Lx8-SINE B2 was recently identified as an ESC-specific lncRNA that marks pluripotency. Here, we studied the function of lncRNA Lx8-SINE B2 in ESCs. Depletion of Lx8-SINE B2 disrupted ESC proliferation, repressed the expression of pluripotency genes, activated differentiation genes, and inhibited reprogramming to induced pluripotent stem cells. The reduction of the colony formation ability of ESCs upon Lx8-SINE B2 knockdown was accompanied by the elongation of the G1 phase and the shortening of the S phase. Transcriptome analysis revealed that Lx8-SINE B2 deficiency affected multiple metabolic pathways, particularly glycolysis. Mechanistically, Lx8-SINE B2 functions as a cytoplasmic lncRNA and interacts with the glycolytic enzyme Eno1 as shown by RNA pull-down and RNA localization analysis. Lx8-SINE B2 and Eno1 interact with and regulate each other's expression, hence promoting the expression of metabolic genes and influencing glycolysis. In conclusion, we have identified lncRNA Lx8-SINE B2 as a novel regulator of ESC proliferation, cell cycle, and metabolism through working with Eno1.

Stem Cell-Based Therapeutic Strategies in Diabetic Wound Healing.

Impaired wound healing and especially the "all-too-common" occurrence of associated diabetic foot ulcers (DFU) are becoming an increasingly urgent and deteriorating healthcare issue, which drastically impact the quality of life and further heighten the risks of infection and amputation in patients with diabetes mellitus. Amongst the multifactorial wound healing determinants, glycemic dysregulation has been identified to be the primary casual factor of poor wound healing. Unfortunately, current therapeutic modalities merely serve as moderate symptomatic relieves but often fail to completely restore the wound site to its pre-injury state and prevent further recurrence. Stem cell-based therapeutics have been employed for its promising potential to address the root of the problem as they not only exhibit the capacity for self-renewal and differentiation towards multiple lineages, but also have been disclosed to participate in mediating variant growth factors and cytokines. Herein we review the current literatures on the therapeutic benefits of using various kinds of stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and adipose-derived stem cells (ASCs) in diabetic wound healing by searching on the PubMed® Database for publications. This study shall serve as an overview of the current body of research with particular focus on autologous ASCs and the laboratory expandable iPSCs in hope of shedding more light on this attractive therapy so as to elevate the efficacy of wound healing that is almost always compromised in diabetic patients.

Regulation of Embryonic Stem Cell Self-Renewal.

Embryonic stem cells (ESCs) are a type of cells capable of self-renewal and multi-directional differentiation. The self-renewal of ESCs is regulated by factors including signaling pathway proteins, transcription factors, epigenetic regulators, cytokines, and small molecular compounds. Similarly, non-coding RNAs, small RNAs, and microRNAs (miRNAs) also play an important role in the process. Functionally, the core transcription factors interact with helper transcription factors to activate the expression of genes that contribute to maintaining pluripotency, while suppressing the expression of differentiation-related genes. Additionally, cytokines such as leukemia suppressor factor (LIF) stimulate downstream signaling pathways and promote self-renewal of ESCs. Particularly, LIF binds to its receptor (LIFR/gp130) to trigger the downstream Jak-Stat3 signaling pathway. BMP4 activates the downstream pathway and acts in combination with Jak-Stat3 to promote pluripotency of ESCs in the absence of serum. In addition, activation of the Wnt-FDZ signaling pathway has been observed to facilitate the self-renewal of ESCs. Small molecule modulator proteins of the pathway mentioned above are widely used in in vitro culture of stem cells. Multiple epigenetic regulators are involved in the maintenance of ESCs self-renewal, making the epigenetic status of ESCs a crucial factor in this process. Similarly, non-coding RNAs and cellular energetics have been described to promote the maintenance of the ESC's self-renewal. These factors regulate the self-renewal and differentiation of ESCs by forming signaling networks. This review focused on the role of major transcription factors, signaling pathways, small molecular compounds, epigenetic regulators, non-coding RNAs, and cellular energetics in ESC's self-renewal.

Multiomics analysis of the NAD+-PARP1 axis reveals a role for site-specific ADP-ribosylation in splicing in embryonic stem cells.

The differentiation of embryonic stem cells (ESCs) into a lineage-committed state is a dynamic process involving changes in cellular metabolism, epigenetic modifications, post-translational modifications, gene expression, and RNA processing. Here we integrated data from metabolomic, proteomic, and transcriptomic assays to characterize how alterations in NAD+ metabolism during the differentiation of mouse ESCs lead to alteration of the PARP1-mediated ADP-ribosylated (ADPRylated) proteome and mRNA isoform specialization. Our metabolomic analyses indicate that mESCs use distinct NAD+ biosynthetic pathways in different cell states: the de novo pathway in the pluripotent state, and the salvage and Preiss-Handler pathways as differentiation progresses. We observed a dramatic induction of PARP1 catalytic activity driven by enhanced nuclear NAD+ biosynthesis during the early stages of mESC differentiation (e.g., within 12 h of LIF removal). PARP1-modified proteins in mESCs are enriched for biological processes related to stem cell maintenance, transcriptional regulation, and RNA processing. The PARP1 substrates include core spliceosome components, such as U2AF35 and U2AF65, whose splicing functions are modulated by PARP1-mediated site-specific ADP-ribosylation. Finally, we observed that splicing is dysregulated genome-wide in Parp1 knockout mESCs. Together, these results demonstrate a role for the NAD+-PARP1 axis in the maintenance of mESC state, specifically in the splicing program during differentiation.

Advances in stem cell therapy in Alzheimer's disease: a comprehensive clinical trial review.

Alzheimer's disease (AD) is the most common type of dementia responsible for more than 121,499 deaths from AD in 2019 making AD the sixth-leading cause in the United States. AD is a progressive neurodegenerative disorder characterized by decline of memory, behavioral impairments that affects a person's ability to function independently ultimately leading to death. The current pressing need for a treatment for (AD) and advances in the field of cell therapy, has rendered stem cell therapeutics a promising field of research. Despite advancements in stem cell technology, confirmed by encouraging pre-clinical utilization of stem cells in AD animal models, the number of clinical trials evaluating the efficacy of stem cell therapy is limited, with the results of many ongoing clinical trials on cell therapy for AD still pending. Mesenchymal stem cells (MSCs) have been the main focus in these studies, reporting encouraging results concerning safety profile, however their efficacy remains unproven. In the current article we review the latest advances regarding different sources of stem cell therapy and present a comprehensive list of every available clinical trial in national and international registries. Finally, we discuss drawbacks arising from AD pathology and technical limitations that hinder the transition of stem cell technology from bench to bedside. Our findings emphasize the need to increase clinical trials towards uncovering the mode of action and the underlying therapeutic mechanisms of transplanted cells as well as the molecular mechanisms controlling regeneration and neuronal microenvironment.

Ectopic Splicing Disturbs the Function of Xist RNA to Establish the Stable Heterochromatin State.

Non-coding Xist RNA plays an essential role in X chromosome inactivation (XCI) in female mammals. It coats the X chromosome in cis and mediates the recruitment of many proteins involved in gene silencing and heterochromatinization. The molecular basis of how Xist RNA initiates chromosomal silencing and what proteins participate in this process has been extensively studied and elucidated. Its involvement in the establishment and maintenance of the X-inactivated state is, however, less understood. The Xist IVS allele we previously reported is peculiar in that it can initiate XCI but fails to establish the inactive state that is stably maintained and, therefore, may provide an opportunity to explore how Xist RNA contributes to establish a robust heterochromatin state. Here we demonstrate that ectopic splicing taking place to produce Xist IVS RNA disturbs its function to properly establish stable XCI state. This finding warrants the potential of Xist IVS RNA to provide further insight into our understanding of how Xist RNA contributes to establish sustainable heterochromatin.

TAF8 regions important for TFIID lobe B assembly or for TAF2 interactions are required for embryonic stem cell survival.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. TFIID is composed of three lobes, named A, B, and C. A 5TAF core complex can be assembled in vitro constituting a building block for the further assembly of either lobe A or B in TFIID. Structural studies showed that TAF8 forms a histone fold pair with TAF10 in lobe B and participates in connecting lobe B to lobe C. To better understand the role of TAF8 in TFIID, we have investigated the requirement of the different regions of TAF8 for the in vitro assembly of lobe B and C and the importance of certain TAF8 regions for mouse embryonic stem cell (ESC) viability. We have identified a region of TAF8 distinct from the histone fold domain important for assembling with the 5TAF core complex in lobe B. We also delineated four more regions of TAF8 each individually required for interacting with TAF2 in lobe C. Moreover, CRISPR/Cas9-mediated gene editing indicated that the 5TAF core-interacting TAF8 domain and the proline-rich domain of TAF8 that interacts with TAF2 are both required for mouse embryonic stem cell survival. Thus, our study defines distinct TAF8 regions involved in connecting TFIID lobe B to lobe C that appear crucial for TFIID function and consequent ESC survival.

Identification of Differentially Expressed Human Endogenous Retrovirus Families in Human Leukemia and Lymphoma Cell Lines and Stem Cells.

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.

Unique Patterns of H3K4me3 and H3K27me3 in 2-Cell-like Embryonic Stem Cells.

A small subgroup of embryonic stem cells (ESCs) exhibit molecular features similar to those of two-cell embryos (2C). However, it remains elusive whether 2C-like cells and 2C embryos share similar epigenetic features. Here, we map the genome-wide profiles of histone H3K4me3 and H3K27me3 in 2C-like cells. We found that the majority of genes in 2C-like cells inherit their histone status from ESCs. Among the genes showing a switch in their histone methylation status during 2C-like transitions, only a small number acquire 2C-embryo epigenetic signatures. In contrast, broad H3K4me3 domains display extensive loss in 2C-like cells. Most of the differentially expressed genes display decreased H3K4me3 and H3K27me3 levels in 2C-like cells, whereas de novo H3K4me3 deposition is closely linked with the expression levels of upregulated 2C-specific genes. Taken together, our study reveals the unique epigenetic profiles of 2C-like cells, facilitating the further exploration of totipotency in the future.