Adult Somatic Stem Cells Research Articles

Prolonging somatic cell proliferation through constitutive hox gene expression in C. elegans

hox genes encode a conserved family of homeodomain transcription factors that are essential to determine the identity of body segments during embryogenesis and maintain adult somatic stem cells competent to regenerate organs. In contrast to higher organisms, somatic cells in C. elegans irreversibly exit the cell cycle after completing their cell lineage and the adult soma cannot regenerate. Here, we show that hox gene expression levels in C. elegans determine the temporal competence of somatic cells to proliferate. Down-regulation of the central hox gene lin-39 in dividing vulval cells results in their premature cell cycle exit, whereas constitutive lin-39 expression causes precocious Pn.p cell and sex myoblast divisions and prolongs the proliferative phase of the vulval cells past their normal point of arrest. Furthermore, ectopic expression of hox genes in the quiescent anchor cell re-activates the cell cycle and induces proliferation until young adulthood. Thus, constitutive expression of a single hox transcription factor is sufficient to prolong somatic cell proliferation beyond the restriction imposed by the cell lineage. The down-regulation of hox gene expression in most somatic cells at the end of larval development may be one cause for the absence of cell proliferation in adult C. elegans.

The Importance of Stem Cells Isolated from Human Dental Pulp and Exfoliated Deciduous Teeth as Therapeutic Approach in Nervous System Pathologies.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for the short-term maintenance of neoblasts invitro and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.

Actin restricts cell proliferation and promotes differentiation during planarian regeneration

Actin is an integral component of the cytoskeleton, which plays an important role in various fundamental cellular processes, such as affecting the polarity of embryonic cells during embryonic development in various model organisms. Meanwhile, previous studies have demonstrated that the polymerization of the actin cytoskeleton can affect cell migration, proliferation, and differentiation. Actin polymerization state regulated osteogenic differentiation and affected cell proliferation. However, the function of actin in regenerative biology has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is an ideal model organism to study regenerative biology. Here, we identified a homolog of actin in planarian Dugesia japonica and found that RNAi targeting actin during planarian regeneration results in the formation of protrusions on the dorsal side, where the division of phospho-H3 mitotic cells is increased. In addition, a decrease in differentiation is observed in regenerating tissues after Djactin RNAi. These results indicate that Djactin functions in proliferation and differentiation control in planarian regeneration.

Human Intestinal Organoids: Promise and Challenge.

The study of human intestinal biology in healthy and diseased conditions has always been challenging. Primary obstacles have included limited tissue accessibility, inadequate in vitro maintenance and ethical constrains. The development of three-dimensional organoid cultures has transformed this entirely. Intestinal organoids are self-organized three-dimensional structures that partially recapitulate the identity, cell heterogeneity and cell behaviour of the original tissue in vitro. This includes the capacity of stem cells to self-renew, as well as to differentiate towards major intestinal lineages. Therefore, over the past decade, the use of human organoid cultures has been instrumental to model human intestinal development, homeostasis, disease, and regeneration. Intestinal organoids can be derived from pluripotent stem cells (PSC) or from adult somatic intestinal stem cells (ISC). Both types of organoid sources harbour their respective strengths and weaknesses. In this mini review, we describe the applications of human intestinal organoids, discussing the differences, advantages, and disadvantages of PSC-derived and ISC-derived organoids.

Computational analysis of spliced leader trans-splicing in the regenerative flatworm Macrostomum lignano reveals its prevalence in conserved and stem cell related genes.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and significantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

Tubgcp3 is a mitotic regulator of planarian epidermal differentiation.

Tubgcp3/GCP3 (The centrosomal protein γ-tubulin complex protein 3) is a component of the γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs), which play critical roles in mitotic spindle formation during mitosis. However, its function in stem cell development has not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is a unique model to study stem cell lineage development in vivo. Here, we identified a homolog of Tubgcp3 in planarian Dugesia japonica, and found that Tubgcp3 is required for the maintenance of epidermal lineage. RNAi targeting Tubgcp3 resulted in tissue homeostasis and regeneration defect. Knockdown of Tubgcp3 reduced cell divisions and led to a loss of the mature epidermal cells. Our findings indicate that Tubgcp3 is a mitotic regulator and plays a crucial role in planarian epidermal differentiation.

5-hydroxymethylcytosine and gene activity in mouse intestinal differentiation

Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.

Understanding the genetics of gastric and esophageal cancer using Drosophila melanogaster as a model organism

Drosophila melanogaster, a vicious model, has helped in significant discoveries about several conserved mechanisms. The insect intestine had remained unexplored until the identification of adult somatic stem cells which triggered the invention of genetic amenability of this insect organ in powerful and artistic ways. A variety of mechanisms within the fruit flies' intestine are conserved in human gastrointestinal systems and will, therefore, become relevant within the context of human pathologies such as gastrointestinal cancers and obesity. In line with current guidelines, maintaining a high degree of suspicion of hereditary etiology (genetic involvement) helps in disease diagnosis. In this review, genetics of two crucial cancers are discussed. Mutation or transcriptional silencing of the cadherin-1 (CDH1) gene and tumor protein p53 gene is related to familial diffuse gastric cancer (GC) and esophageal adenocarcinoma, respectively. Testing for CDH1 mutations in patients with familial clustering of hereditary diffuse GC has proven consistent. Further studies on the expression and also the alteration within the proteins within the E-CDH pathways and Hippo pathway may function as biomarkers for early detection. Dysregulation of various mechanisms ends up in invasion, proliferation, and metastases. The promise of targeted therapy and personalized medicine in improving the clinical outcome is now closer than it has ever been.

Region-specific regulation of stem cell-driven regeneration in tapeworms.

Tapeworms grow at rates rivaling the fastest-growing metazoan tissues. To propagate they shed large parts of their body; to replace these lost tissues they regenerate proglottids (segments) as part of normal homeostasis. Their remarkable growth and regeneration are fueled by adult somatic stem cells that have yet to be characterized molecularly. Using the rat intestinal tapeworm, Hymenolepis diminuta, we find that regenerative potential is regionally limited to the neck, where head-dependent extrinsic signals create a permissive microenvironment for stem cell-driven regeneration. Using transcriptomic analyses and RNA interference, we characterize and functionally validate regulators of tapeworm growth and regeneration. We find no evidence that stem cells are restricted to the regeneration-competent neck. Instead, lethally irradiated tapeworms can be rescued when cells from either regeneration-competent or regeneration-incompetent regions are transplanted into the neck. Together, the head and neck tissues provide extrinsic cues that regulate stem cells, enabling region-specific regeneration in this parasite.

Choroid plexus-derived miR-204 regulates the number of quiescent neural stem cells in the adult brain.

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.

Cdc42 regulate the apoptotic cell death required for planarian epidermal regeneration and homeostasis.

Rho GTPases have been shown previously to play important roles in several cellular processes by regulating the organization of the actin and microtubule cytoskeletons. However, the mechanisms of Rho GTPases that integrate the cellular responses during regeneration have not been thoroughly elucidated. The planarian flatworm, which contains a large number of adult somatic stem cells (neoblasts), is a unique model to study stem cell lineage development in vivo. Here, we focus on cdc42, which is an extensively characterized member among Rho GTPases. We found that cdc42 is required for the maintenance of epidermal lineage. Cdc42 RNAi induced a sustained increased of cell death and led to a loss of the mature epidermal cells but without affected cell division. Our results indicate that cdc42 function as an inhibitor to block the excessive apoptotic cell death in planarian epidermal regeneration and homeostasis.

An Overview About the Biology of Skeletal Muscle Satellite Cells

The peculiar ability of skeletal muscle tissue to operate adaptive changes during post-natal de-velopment and adulthood has been associated with the existence of adult somatic stem cells. Satellite cells, occupying an exclusive niche within the adult muscle tissue, are considered bona fide stem cells with both stem-like properties and myogenic activities. Indeed, satellite cells retain the capability to both maintain the quiescence in uninjured muscles and to be promptly activated in response to growth or re-generative signals, re-engaging the cell cycle. Activated cells can undergo myogenic differentiation or self-renewal moving back to the quiescent state. Satellite cells behavior and their fate decision are finely controlled by mechanisms involving both cell-autonomous and external stimuli. Alterations in these regu-latory networks profoundly affect muscle homeostasis and the dynamic response to tissue damage, con-tributing to the decline of skeletal muscle that occurs under physio-pathologic conditions. Although the clear myogenic activity of satellite cells has been described and their pivotal role in muscle growth and regeneration has been reported, a comprehensive picture of inter-related mechanisms guiding muscle stem cell activity has still to be defined. Here, we reviewed the main regulatory networks determining satellite cell behavior. In particular, we focused on genetic and epigenetic mechanisms underlining satel-lite cell maintenance and commitment. Besides intrinsic regulations, we reported current evidences about the influence of environmental stimuli, derived from other cell populations within muscle tissue, on satel-lite cell biology.

Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Keratinocyte Precursor Cells.

The functional definition of somatic adult stem cells is based on their regenerative capacity, which allows tissue regeneration throughout life. Thus, refining methodologies to characterize this capacity is of great importance for progress in the fundamental knowledge of specific keratinocyte subpopulations but also for preclinical and clinical research, considering the high potential of keratinocytes in cell therapy. We present here a methodology which we define as iterative xenografting, which originates in the classical model of human skin substitute xenografts onto immunodeficient recipient mice. The principle of this functional assay is first to perform primary xenografts to assess graft take and the quality of epidermal differentiation. Then, human keratinocytes are extracted from primary graft samples to perform secondary xenografts, to assess the presence and preservation of functional keratinocyte stem cells with long-term regenerative potential. In the example of experiments shown, iterative skin xenografting was used to document the high regenerative potential of epidermal holoclone keratinocytes.

Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

To compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) of Drosophila and required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by the Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosis DIAP1. These data therefore identify Svb as a nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

Anatomy and Physiology of the Digestive Tract of Drosophila melanogaster.

The gastrointestinal tract has recently come to the forefront of multiple research fields. It is now recognized as a major source of signals modulating food intake, insulin secretion and energy balance. It is also a key player in immunity and, through its interaction with microbiota, can shape our physiology and behavior in complex and sometimes unexpected ways. The insect intestine had remained, by comparison, relatively unexplored until the identification of adult somatic stem cells in the Drosophila intestine over a decade ago. Since then, a growing scientific community has exploited the genetic amenability of this insect organ in powerful and creative ways. By doing so, we have shed light on a broad range of biological questions revolving around stem cells and their niches, interorgan signaling and immunity. Despite their relatively recent discovery, some of the mechanisms active in the intestine of flies have already been shown to be more widely applicable to other gastrointestinal systems, and may therefore become relevant in the context of human pathologies such as gastrointestinal cancers, aging, or obesity. This review summarizes our current knowledge of both the formation and function of the Drosophila melanogaster digestive tract, with a major focus on its main digestive/absorptive portion: the strikingly adaptable adult midgut.

Direct Reprogramming of Adult Human Somatic Stem Cells Into Functional Neurons Using Sox2, Ascl1, and Neurog2.

Reprogramming of somatic cells into induced pluripotent stem cells (iPS) or directly into cells from a different lineage, including neurons, has revolutionized research in regenerative medicine in recent years. Mesenchymal stem cells are good candidates for lineage reprogramming and autologous transplantation, since they can be easily isolated from accessible sources in adult humans, such as bone marrow and dental tissues. Here, we demonstrate that expression of the transcription factors (TFs) SRY (sex determining region Y)-box 2 (Sox2), Mammalian achaete-scute homolog 1 (Ascl1), or Neurogenin 2 (Neurog2) is sufficient for reprogramming human umbilical cord mesenchymal stem cells (hUCMSC) into induced neurons (iNs). Furthermore, the combination of Sox2/Ascl1 or Sox2/Neurog2 is sufficient to reprogram up to 50% of transfected hUCMSCs into iNs showing electrical properties of mature neurons and establishing synaptic contacts with co-culture primary neurons. Finally, we show evidence supporting the notion that different combinations of TFs (Sox2/Ascl1 and Sox2/Neurog2) may induce multiple and overlapping neuronal phenotypes in lineage-reprogrammed iNs, suggesting that neuronal fate is determined by a combination of signals involving the TFs used for reprogramming but also the internal state of the converted cell. Altogether, the data presented here contribute to the advancement of techniques aiming at obtaining specific neuronal phenotypes from lineage-converted human somatic cells to treat neurological disorders.

Molecular Reproduction & Development Volume 84, Issue 12, December 2017

The planarian flatworm Schmidtea mediterranea develops its entire reproductive system post‐embryonically from a population of adult somatic stem cells. Counts et al. (this issue) assessed the requirement of different chaperonin CCT/TRiC subunits during spermatogenesis and somatic integrity using RNA‐interference, which allows for the genetic assessment of specific components during oogenesis and spermatogenesis in this organism. Shown here is a sexually mature planarian (S. mediterranea), imaged by dark‐field microscopy, overlaid on a confocal image of the interior of an individual testis lobe in which the nuclei of cells during various stages of spermatogenesis are revealed.

Limbal Stem Cell Transplantation and Complications

ABSTRACT Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).