Population Of Pluripotent Stem Cells Research Articles

Alterations in Endogenous Stem Cell Populations in the Acute Phase of Blast-Induced Spinal Cord Injury.

Blast-induced spinal cord injury (bSCI) is prevalent among military populations and frequently leads to irreversible spinal cord tissue damage that manifests as sensorimotor and autonomic nervous system dysfunction. Clinical recovery from bSCI has been proven to be multifactorial, as it is heavily dependent on the function of numerous cell populations in the tissue environment, as well as extensive ongoing inflammatory processes. This varied recovery process is thought to be due to irreversible spinal cord damage after 72 hours post-injury. Stem cell therapy for spinal cord injuries has long been investigated due to these cells' proliferative nature, ability to enhance neuro-regeneration, neuroprotection, remyelination of axons, and modulation of the immune and inflammatory responses. Therefore, this study hypothesizes that the impaired function after injury is due to a lack of specific ectoderm and neural stem cell population activity at the injury site. This study aimed to elucidate changes in endogenous stem cell patterns by evaluating immunohistochemical staining densities of various stem cell markers using a preclinical thoracolumbar bSCI model. Analysis was performed 24-, 48-, and 72 hours following blast exposure. Behavior tests to assess sensory and mechanical functions were also performed. The following Cluster of differentiation (CD) markers CD105, CD45, CD133, and Vimentin, Nanog homebox (NANOG), and sex determining region Y HMG-box 2 (SOX2) positive cell populations were significantly elevated with trending increases in Octamer-binding transcription factor 4 (OCT4) in the thoracolumbar region of spinal cord tissue at 72 hours following bSCI (p < 0.05). Behavior analyses showed significant decreases in paw withdrawal thresholds in the hind limbs and changes in locomotion at 48- and 72 hours post-injury (p < 0.05). The significant increase in mesenchymal, pluripotent, and neural stem cell populations within the thoracolumbar region post-injury suggests that migratory patterns of stem cell populations are likely altered in response to bSCI. Behavioral deficits were consistent with those experienced by military personnel, such as increased pain-like behavior, reduced proprioception and coordination, and increased anxiety-like behavior post-bSCI, which underlines the translational capabilities of this model. While further research is vital to understand better the intrinsic and synergistic chemical and mechanical factors driving the migration of stem cells after traumatic injury, increased endogenous stem cell populations at the injury site indicate that stem cell-based treatments in patients suffering from bSCI could prove beneficial.

Starvation resistance in planarians: multiple strategies to get a thrifty phenotype.

Starvation resistance is a life-saving mechanism for many organisms facing food availability fluctuation in the natural environment. Different strategies have been episodically identified for some model organisms, the first of which was the ability to suppress metabolic rate. Among the identified strategies, the ability of planarians to shrink their body under fasting conditions and revert the process after feeding (the growth-degrowth process) represents a fascinating mechanism to face long periods of fasting. The growth-degrowth process is strictly related to the capability of planarians to continuously maintain tissue homeostasis and body proportions even in challenging conditions, thanks to the presence of a population of pluripotent stem cells. Here, we take advantage of several previous studies describing the growth-degrowth process and of recent progress in the understanding of planarian homeostasis mechanisms, to identify tissue-selective transcriptional downregulation as a driving strategy for the development of a thrifty phenotype, and the p53 transcription factor as a player in adjusting tissue homeostasis in accordance with food availability.

Stem Cells: Engines of Plant Growth and Development.

The development of both animals and plants relies on populations of pluripotent stem cells that provide the cellular raw materials for organ and tissue formation. Plant stem cell reservoirs are housed at the shoot and root tips in structures called meristems, with the shoot apical meristem (SAM) continuously producing aerial leaf, stem, and flower organs throughout the life cycle. Thus, the SAM acts as the engine of plant development and has unique structural and molecular features that allow it to balance self-renewal with differentiation and act as a constant source of new cells for organogenesis while simultaneously maintaining a stem cell reservoir for future organ formation. Studies have identified key roles for intercellular regulatory networks that establish and maintain meristem activity, including the KNOX transcription factor pathway and the CLV-WUS stem cell feedback loop. In addition, the plant hormones cytokinin and auxin act through their downstream signaling pathways in the SAM to integrate stem cell activity and organ initiation. This review discusses how the various regulatory pathways collectively orchestrate SAM function and touches on how their manipulation can alter stem cell activity to improve crop yield.

Acoel single-cell atlas reveals expression dynamics and heterogeneity of adult pluripotent stem cells

Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.

Pluripotent stem cells: Basic biology or else differentiations aimed at translational research and the role of flow cytometry.

Pluripotent stem cell research has revolutionized the modern era for the past 14 years with the advent of induced pluripotent stem cells. Before this time, scientists had access to human and mouse embryonic stem cells primarily for basic research and an attempt towards lineage-specific differentiations for cell therapy applications. Regarding pluripotent stem cells, expression of bonafide marker proteins such as Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 have been considered giving a perfect readout for pluripotent stem cells and assessed using an analytical flow cytometer. In addition to the intracellular markers, surface markers such as stage-specific embryonic antigen-1 for mouse cells and SSEA-4 for human cells are needed to sort pure populations of stem cells for further downstream applications for cell therapy. The surface marker SSEA-4 is the most appropriate for obtaining pure populations of human pluripotent stem cells. When differentiated in a controlled manner using growth factors or small molecules, it is mandatory to assess the downregulation of pluripotency markers (Oct4, Nanog, Sox2, and Klf4) with subsequent up-regulation of stage-specific differentiation markers. Such assessments are done using flow cytometry. Pluripotent stem cells have a high teratoma-forming potential in vivo. Small amounts of undifferentiated PSCs might lead to dangerous teratomas upon transplantation if leftover in the pool of differentiated cells. Hence, flow cytometry is essential for sorting out PSC populations with teratoma-forming potential. The pure populations of differentiated progenitors need to be flow-sorted before differentiating them further for cell therapy applications. For example, Glycoprotein 2 is a specific cell-surface marker for pancreatic progenitors that enables one to sort the pancreatic progenitors differentiated from human PSCs. Taken together, analytical flow cytometry, and cell sorting provide indispensable tools in PSC research and cell therapy.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

GFP Tagged VSELs Help Delineate Novel Stem Cells Biology in Multiple Adult Tissues.

Various types of stem cells are being researched upon to exploit their potential for regenerative medicine including pluripotent human embryonic stem (hES) cells derived from spare human embryos, induced pluripotent stem (iPS) cells by reprogramming somatic cells to a pluripotent state and multipotent mesenchymal stem/stromal cells (MSCs) obtained in vitro from multiple tissues. More than 50 independent groups have reported another novel population of pluripotent stem cells in adult tissues termed very small embryonic-like stem cells (VSELs). VSELs are developmentally linked to primordial germ cells, which rather than giving rise to the germ cells and later ceasing to exist, survive throughout life in multiple organs along with tissue-specific adult stem cells better described as lineage-restricted, tissue-committed progenitors with limited plasticity. VSELs survive total body irradiation in bone marrow, oncotherapy in the gonads, bilateral ovariectomy in the uterus and partial pancreatectomy in the pancreas of mice and participate in the regeneration of multiple organs under normal physiological conditions. VSELs and tissue-specific progenitor cells work together in a subtle manner, maintain life-long tissue homeostasis and their dysfunction leads to various pathologies including cancer. However, due to their quiescent state, VSELs have invariably eluded lineage-tracing studies reported so far. Present article reviews novel insights into VSELs biology and how VSELs enriched from GFP (green fluorescent protein) mice have enabled to delineate their role in various biological processes in vivo. VSELs biology needs to be understood in-depth as this alone will help evolve the field of regenerative medicine and win the war against cancer.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Generation and characterization of hair-bearing skin organoids from human pluripotent stem cells.

Human skin uses millions of hairs and glands distributed across the body surface to function as an external barrier, thermoregulator and stimuli sensor. The large-scale generation of human skin with these appendages would be beneficial, but is challenging. Here, we describe a detailed protocol for generating hair-bearing skin tissue entirely from a homogeneous population of human pluripotent stem cells in a three-dimensional in vitro culture system. Defined culture conditions are used over a 2-week period to induce differentiation of pluripotent stem cells to surface ectoderm and cranial neural crest cells, which give rise to the epidermis and dermis, respectively, in each organoid unit. After 60 d of incubation, the skin organoids produce hair follicles. By day ~130, the skin organoids reach full complexity and contain stratified skin layers, pigmented hair follicles, sebaceous glands, Merkel cells and sensory neurons, recapitulating the cell composition and architecture of fetal skin tissue at week 18 of gestation. Skin organoids can be maintained in culture using this protocol for up to 150 d, enabling the organoids to be used to investigate basic skin biology, model disease and, further, reconstruct or regenerate skin tissue.

FoxK1 is Required for Ectodermal Cell Differentiation During Planarian Regeneration.

Forkhead box (Fox) genes belong to the “winged helix” transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues.

Home Away From Home: Bioengineering Advancements to Mimic the Developmental and Adult Stem Cell Niche

The inherent self-organizing capacity of pluripotent and adult stem cell populations has advanced our fundamental understanding of processes that drive human development, homeostasis, regeneration, and disease progression. Translating these principles into in vitro model systems has been achieved with the advent of organoid technology, driving innovation to harness patient-specific, cell-laden regenerative constructs that can be engineered to augment or replace diseased tissue. While developmental organization and regenerative adult stem cell niches are tightly regulated in vivo, in vitro analogs lack defined architecture and presentation of physicochemical cues, leading to the unhindered arrangement of mini-tissues that lack complete physiological mimicry. This review aims to highlight the recent integrative engineering approaches that elicit spatio-temporal control of the extracellular niche to direct the structural and functional maturation of pluripotent and adult stem cell derivatives. While the advances presented here leverage multi-pronged strategies ranging from synthetic biology to microfabrication technologies, the methods converge on recreating the biochemical and biophysical milieu of the native tissue to be modeled or regenerated.

Loss of plac8 expression rapidly leads pluripotent stem cells to enter active state during planarian regeneration.

The regenerative ability of planarians relies on their adult pluripotent stem cell population. Although all stem cells express a piwi homolog, recently it has become possible to classify the piwi+ stem cell population into specialized subpopulations according to the expression of genes related to differentiation. However, piwi+ stem cells behave practically as a homogeneous population after amputation, during which stem cells show accelerated proliferation, named 'induced hyperproliferation'. Here, we show that plac8-A was expressed in almost all of the stem cells, and that a decrease of the plac8-A expression level led to induced hyperproliferation uniformly in a broad stem cell subpopulation after amputation. This reduction of plac8-A expression was caused by activated JNK signaling after amputation. Pharmacological inhibition of JNK signaling caused failure to induce hyperproliferation and resulted in regenerative defects. Such defects were abrogated by simultaneous knockdown of plac8-A expression. Thus, JNK-dependent suppression of plac8-A expression is indispensable for stem cell dynamics involved in regeneration. These findings suggest that plac8-A acts as a molecular switch of piwi+ stem cells for entry into the regenerative state after amputation.

RNAi Screening to Assess Tissue Regeneration in Planarians.

Over the past several decades, planarians have emerged as a powerful model system with which to study the cellular and molecular basis of whole-body regeneration. The best studied planarians belong to freshwater flatworm species that maintain their remarkable regenerative capacity partly through the deployment of a population of adult pluripotent stem cells. Assessment of gene function in planarian regeneration has primarily been achieved through RNA interference (RNAi), either through the feeding or injection of double-stranded RNA (dsRNA). RNAi treatment of planarians has several advantages, including ease of use, which allows for medium-throughput screens of hundreds of genes over the course of a single project. Here, I present methods for dsRNA synthesis and RNAi feeding, as well as strategies for follow-up assessment of both structural and functional regeneration of organ systems of planarians, with a special emphasis on neural regeneration.

Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells.

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

Decoding Stem Cells: An Overview on Planarian Stem Cell Heterogeneity and Lineage Progression.

Planarians are flatworms capable of whole-body regeneration, able to regrow any missing body part after injury or amputation. The extraordinary regenerative capacity of planarians is based upon the presence in the adult of a large population of somatic pluripotent stem cells. These cells, called neoblasts, offer a unique system to study the process of stem cell specification and differentiation in vivo. In recent years, FACS-based isolation of neoblasts, RNAi functional analyses as well as high-throughput approaches such as single-cell sequencing have allowed a rapid progress in our understanding of many different aspects of neoblast biology. Here, we summarize our current knowledge on the molecular signatures that define planarian neoblasts heterogeneity, which includes a percentage of truly pluripotent stem cells, and guide the commitment of pluripotent neoblasts into lineage-specific progenitor cells, as well as their differentiation into specific planarian cell types.

A case for revisiting Nodal signaling in human pluripotent stem cells.

Nodal is a transforming growth factor-β (TGF-β) superfamily member that plays a number of critical roles in mammalian embryonic development. Nodal is essential for the support of the peri-implantation epiblast in the mouse embryo and subsequently acts to specify mesendodermal fate at the time of gastrulation and, later, left-right asymmetry. Maintenance of human pluripotent stem cells (hPSCs) in vitro is dependent on Nodal signaling. Because it has proven difficult to prepare a biologically active form of recombinant Nodal protein, Activin or TGFB1 are widely used as surrogates for NODAL in hPSC culture. Nonetheless, the expression of the components of an endogenous Nodal signaling pathway in hPSC provides a potential autocrine pathway for the regulation of self-renewal in this system. Here we review recent studies that have clarified the role of Nodal signaling in pluripotent stem cell populations, highlighted spatial restrictions on Nodal signaling, and shown that Nodal functions in vivo as a heterodimer with GDF3, another TGF-β superfamily member expressed by hPSC. We discuss the role of this pathway in the maintenance of the epiblast and hPSC in light of these new advances.

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

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

Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies.

Human pluripotent stem cells (PSCs), which are composed of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an opportunity to advance cardiac cell therapy–based clinical trials. However, an important hurdle that must be overcome is the risk of teratoma formation after cell transplantation due to the proliferative capacity of residual undifferentiated PSCs in differentiation batches. To tackle this problem, we propose the use of a minimal noncardiotoxic doxorubicin dose as a purifying agent to selectively target rapidly proliferating stem cells for cell death, which will provide a purer population of terminally differentiated cardiomyocytes before cell transplantation. In this study, we determined an appropriate in vitro doxorubicin dose that (a) eliminates residual undifferentiated stem cells before cell injection to prevent teratoma formation after cell transplantation and (b) does not cause cardiotoxicity in ESC-derived cardiomyocytes (CMs) as demonstrated through contractility analysis, electrophysiology, topoisomerase activity assay, and quantification of reactive oxygen species generation. This study establishes a potentially novel method for tumorigenic-free cell therapy studies aimed at clinical applications of cardiac cell transplantation.

Pluripotent stem cells in the research for extraembryonic cell differentiation.

Mouse embryonic stem cells (mESCs) are pluripotent stem cell populations derived from the preimplantation embryo and are used to study the differentiation of many types of somatic and germ cells in developing embryos. They are also used to study cell lineages of extraembryonic tissues, such as the trophectoderm (TE) and the primitive endoderm (PrE). mESC cultures are suitable systems for reproducing cellular and molecular events occurring during the differentiation of these cell types, such as changes in gene expression patterns, signaling events, and genome rearrangements although the consistency between the results obtained using mESCs and those of in vivo studies on embryos should be carefully taken into account. Since TE and PrE cells can be induced from mESCs in vitro, mESC cultures are useful systems to study differentiation of these cell lineages during development, if used appropriately. In addition, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), are capable of generating extraembryonic lineages in vitro and are promising tools to study the differentiation of these lineages in the human embryo.

Plant stem-cell organization and differentiation at single-cell resolution

Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing (scRNA-seq) to achieve an unbiased characterization of the transcriptional landscape of the maize shoot stem-cell niche and its differentiating cellular descendants. Stem cells housed in the SAM tip are engaged in genome integrity maintenance and exhibit a low rate of cell division, consistent with their contributions to germline and somatic cell fates. Surprisingly, we find no evidence for a canonical stem-cell organizing center subtending these cells. In addition, trajectory inference was used to trace the gene expression changes that accompany cell differentiation, revealing that ectopic expression of KNOTTED1 (KN1) accelerates cell differentiation and promotes development of the sheathing maize leaf base. These single-cell transcriptomic analyses of the shoot apex yield insight into the processes of stem-cell function and cell-fate acquisition in the maize seedling and provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis at the cellular level.