PI3K Plays an Essential Role in Planarian Regeneration and Tissue Maintenance

Phosphatidylinositol 3-kinase (PI3K) signaling plays a central role in various biological processes, and its abnormality leads to a broad spectrum of human diseases, such as cancer, fibrosis, and immunological disorders. However, the mechanisms by which PI3K signaling regulates the behavior of stem cells during regeneration are poorly understood. Planarian flatworms possess abundant adult stem cells (called neoblasts) allowing them to develop remarkable regenerative capabilities, thus the animals represent an ideal model for studying stem cells and regenerative medicine in vivo. In this study, the spatiotemporal expression pattern of Djpi3k, a PI3K ortholog in the planarian Dugesia japonica, was investigated and suggests its potential role in wound response and tissue regeneration. A loss-of-function study was conducted using small molecules and RNA interference technique, providing evidence that PI3K signaling is required for blastema regrowth and cilia maintenance during planarian regeneration and homeostasis. Interestingly, the mitotic and apoptotic responses to amputation are substantially abated in PI3K inhibitor-treated regenerating animals, while knockdown of Djpi3k alleviates the mitotic response and postpones the peak of apoptotic cell death, which may contribute to the varying degrees of regenerative defects induced by the pharmacological and genetic approaches. These observations reveal novel roles for PI3K signaling in the regulation of the cellular responses to amputation during planarian regeneration and provide insights for investigating the disease-related genes in the regeneration-competent organism in vivo.

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The Act of Controlling Adult Stem Cell Dynamics: Insights from Animal Models

Biomolecules ◽

10.3390/biom11050667 ◽

2021 ◽

Vol 11 (5) ◽

pp. 667

Author(s):

Meera Krishnan ◽

Sahil Kumar ◽

Luis Johnson Kangale ◽

Eric Ghigo ◽

Prasad Abnave

Keyword(s):

Stem Cells ◽

Animal Models ◽

Adult Stem Cells ◽

The Body ◽

In Vivo Studies ◽

Self Renewal ◽

Hematopoietic Stem ◽

Organ Systems

Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs).

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Organoid based personalized medicine: from bench to bedside

Cell Regeneration ◽

10.1186/s13619-020-00059-z ◽

2020 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Yaqi Li ◽

Peiyuan Tang ◽

Sanjun Cai ◽

Junjie Peng ◽

Guoqiang Hua

Keyword(s):

Stem Cells ◽

Personalized Medicine ◽

Adult Stem Cells ◽

Three Dimensional ◽

Basic Research ◽

Normal Tissues ◽

Technology Applications ◽

Genetically Manipulated

AbstractThree-dimensional cultured organoids have become a powerful in vitro research tool that preserves genetic, phenotypic and behavioral trait of in vivo organs, which can be established from both pluripotent stem cells and adult stem cells. Organoids derived from adult stem cells can be established directly from diseased epithelium and matched normal tissues, and organoids can also be genetically manipulated by CRISPR-Cas9 technology. Applications of organoids in basic research involve the modeling of human development and diseases, including genetic, infectious and malignant diseases. Importantly, accumulating evidence suggests that biobanks of patient-derived organoids for many cancers and cystic fibrosis have great value for drug development and personalized medicine. In addition, organoids hold promise for regenerative medicine. In the present review, we discuss the applications of organoids in the basic and translational research.

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Mesenchymal stem cell-derived exosomal microRNA-136-5p inhibits chondrocyte degeneration in traumatic osteoarthritis by targeting ELF3

Arthritis Research & Therapy ◽

10.1186/s13075-020-02325-6 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Xue Chen ◽

Yuanyuan Shi ◽

Pan Xue ◽

Xinli Ma ◽

Junfeng Li ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Mouse Model ◽

Cartilage Degeneration ◽

Clinical Samples ◽

Loss Of Function ◽

Post Traumatic ◽

Chondrocyte Migration

Abstract Background Emerging evidence suggests that microRNAs (miRs) are associated with the progression of osteoarthritis (OA). In this study, the role of exosomal miR-136-5p derived from mesenchymal stem cells (MSCs) in OA progression is investigated and the potential therapeutic mechanism explored. Methods Bone marrow mesenchymal stem cells (BMMSCs) and their exosomes were isolated from patients and identified. The endocytosis of chondrocytes and the effects of exosome miR-136-5p on cartilage degradation were observed and examined by immunofluorescence and cartilage staining. Then, the targeting relationship between miR-136-5p and E74-like factor 3 (ELF3) was analyzed by dual-luciferase report assay. Based on gain- or loss-of-function experiments, the effects of exosomes and exosomal miR-136-5p on chondrocyte migration were examined by EdU and Transwell assay. Finally, a mouse model of post-traumatic OA was developed to evaluate effects of miR-136-5p on chondrocyte degeneration in vivo. Results In the clinical samples of traumatic OA cartilage tissues, we detected increased ELF3 expression, and reduced miR-136-5p expression was determined. The BMMSC-derived exosomes showed an enriched level of miR-136-5p, which could be internalized by chondrocytes. The migration of chondrocyte was promoted by miR-136-5p, while collagen II, aggrecan, and SOX9 expression was increased and MMP-13 expression was reduced. miR-136-5p was verified to target ELF3 and could downregulate its expression. Moreover, the expression of ELF3 was reduced in chondrocytes after internalization of exosomes. In the mouse model of post-traumatic OA, exosomal miR-136-5p was found to reduce the degeneration of cartilage extracellular matrix. Conclusion These data provide evidence that BMMSC-derived exosomal miR-136-5p could promote chondrocyte migration in vitro and inhibit cartilage degeneration in vivo, thereby inhibiting OA pathology, which highlighted the transfer of exosomal miR-136-5p as a promising therapeutic strategy for patients with OA.

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Alternative polyadenylation of Pax3 controls muscle stem cell fate and muscle function

Science ◽

10.1126/science.aax1694 ◽

2019 ◽

Vol 366 (6466) ◽

pp. 734-738 ◽

Cited By ~ 11

Author(s):

Antoine de Morree ◽

Julian D. D. Klein ◽

Qiang Gan ◽

Jean Farup ◽

Andoni Urtasun ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Messenger Rna ◽

Adult Stem Cells ◽

Alternative Polyadenylation ◽

Quiescent State ◽

Stem Cell Fate ◽

Activation Rate

Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3′ untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.

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Implications and Current Limitations of Oogenesis from Female Germline or Oogonial Stem Cells in Adult Mammalian Ovaries

Cells ◽

10.3390/cells8020093 ◽

2019 ◽

Vol 8 (2) ◽

pp. 93 ◽

Cited By ~ 16

Author(s):

Jessica Martin ◽

Dori Woods ◽

Jonathan Tilly

Keyword(s):

Stem Cells ◽

Adult Stem Cells ◽

Ovarian Function ◽

Mammalian Species ◽

Large Body ◽

The Body ◽

Human Female ◽

Female Germline

A now large body of evidence supports the existence of mitotically active germ cells in postnatal ovaries of diverse mammalian species, including humans. This opens the possibility that adult stem cells naturally committed to a germline fate could be leveraged for the production of female gametes outside of the body. The functional properties of these cells, referred to as female germline or oogonial stem cells (OSCs), in ovaries of women have recently been tested in various ways, including a very recent investigation of the differentiation capacity of human OSCs at a single cell level. The exciting insights gained from these experiments, coupled with other data derived from intraovarian transplantation and genetic tracing analyses in animal models that have established the capacity of OSCs to generate healthy eggs, embryos and offspring, should drive constructive discussions in this relatively new field to further exploring the value of these cells to the study, and potential management, of human female fertility. Here, we provide a brief history of the discovery and characterization of OSCs in mammals, as well as of the in-vivo significance of postnatal oogenesis to adult ovarian function. We then highlight several key observations made recently on the biology of OSCs, and integrate this information into a broader discussion of the potential value and limitations of these adult stem cells to achieving a greater understanding of human female gametogenesis in vivo and in vitro.

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In Vivo Tracking of Murine Adipose Tissue-Derived Multipotent Adult Stem Cells and Ex Vivo Cross-Validation

International Journal of Molecular Imaging ◽

10.1155/2013/426961 ◽

2013 ◽

Vol 2013 ◽

pp. 1-13 ◽

Cited By ~ 9

Author(s):

Chiara Garrovo ◽

Natascha Bergamin ◽

Dave Bates ◽

Daniela Cesselli ◽

Antonio Paolo Beltrami ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Optical Imaging ◽

Adult Stem Cells ◽

Near Infrared ◽

Cross Validation ◽

Ex Vivo ◽

Imaging Data ◽

Imaging Results

Stem cells are characterized by the ability to renew themselves and to differentiate into specialized cell types, while stem cell therapy is believed to treat a number of different human diseases through either cell regeneration or paracrine effects. Herein, an in vivo and ex vivo near infrared time domain (NIR TD) optical imaging study was undertaken to evaluate the migratory ability of murine adipose tissue-derived multipotent adult stem cells [mAT-MASC] after intramuscular injection in mice. In vivo NIR TD optical imaging data analysis showed a migration of DiD-labelled mAT-MASC in the leg opposite the injection site, which was confirmed by a fibered confocal microendoscopy system. Ex vivo NIR TD optical imaging results showed a systemic distribution of labelled cells. Considering a potential microenvironmental contamination, a cross-validation study by multimodality approaches was followed: mAT-MASC were isolated from male mice expressing constitutively eGFP, which was detectable using techniques of immunofluorescence and qPCR. Y-chromosome positive cells, injected into wild-type female recipients, were detected by FISH. Cross-validation confirmed the data obtained by in vivo/ex vivo TD optical imaging analysis. In summary, our data demonstrates the usefulness of NIR TD optical imaging in tracking delivered cells, giving insights into the migratory properties of the injected cells.

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Phenotypical, Morphological and Molecular Analysis of Fresh Human Bone Marrow Mesenchymal/Stromal Stem Sells (MSCs) Enriched by Four Different Methods.

Blood ◽

10.1182/blood.v104.11.2337.2337 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2337-2337

Author(s):

Elena A. Jones ◽

Frederique Ponchel ◽

Sally E. Kinsey ◽

Sarah L. Field ◽

Liz Straszynski ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Adult Stem Cells ◽

Magnetic Beads ◽

High Specificity ◽

Human Bone ◽

Human Bone Marrow ◽

Older Individuals ◽

Bmp Receptors

Abstract Methods have recently been described for purification of mesenchymal stem cells (MSCs) from human bone marrow (BM), based on different combinations of antibodies and magnetic beads (Quirici et al, 2002, Reyes et al, 2002, Jones et al, 2002, Gronthos et al, 2003). We have previously demonstrated that BM MSCs reside in the CD45lowD7-FIB+LNGFR+ cell fraction (Jones et al, 2002). In the present work we compared four different microbead-based enrichment methods and showed that positive selection with anti-fibroblast (D7-FIB) microbeads (n=28), LNGFR microbeads (n=5) and plastic adherence/trypsinization (n=6) resulted in similar enrichment of cells with this phenotype, with their frequency rising from 0.01% in un-fractionated marrow to 6.6±5.8%, 7.7±7.0 and 4.9±1.5%, respectively, in the enriched fraction. Although MSC isolation by negative depletion with CD45 and GPA microbeads produced poorer enrichment (1.3±0.9%, n=5) its advantage was that MSCs remained unlabelled. Defining the best positive marker for selection requires its high expression on MSCs (high specificity) and lack of binding to CD45+ cells (high selectivity). LNGFR remained the best marker, followed by CD146, CD106, D7-FIB, CD13 and CD166. In contrast, CD105, CD73, CD44 and CD63 were not very selective and showed only moderate levels of expression on MSCs. Mesenchymal nature of sorted CD45lowLNGFR+ cells was further confirmed by real-time PCR measurements of transcription factors (TFs) implicated in mesenchymal lineage differentiation towards bone, cartilage and adipose tissue. Compared with matched haematopoietic lineage cells (CD45+ cells) freshly purified CD45lowLNGFR+ MSCs showed significantly higher expression of cbfa-1, Sox9 and PPARγ ranging from 20- to 150-fold. IL-7 RNA was also 60-fold more abundant in sorted MSCs than in CD45+ cells. Whether these TFs and IL-7 were expressed by the same cells or in separate populations of osteoprogenitors and preadipocytes, remains to be established. Oil red staining of individual, freshly sorted MSCs, however, revealed that some cells contained microscopic fat droplets and thus were already engaged in adipogenic progression, particularly in older individuals. Sox9 and cbfa-1 expression levels were similar in freshly purified and expanded MSCs, whilst the expression of PPARγ and IL-7 declined during standard expansion, but could be restored in adipogenic and stromal media, respectively. In vivo levels of stromal differentiation and adipogenic progression therefore appear to be transiently lost following standard MSC expansion. Whilst adipogenesis seems to be environmentally regulated, osteogenesis, on the other hand, may indeed be a default state of MSCs in the BM. Consistent with this idea, osteogenesis-related markers (alkaline phospatase, and BMP receptors) were uniformly, and not differentially, expressed by freshly sorted MSCs. Finally, freshly purified MSCs did not express ES-cell markers SSEA-3, SSEA-4, and Tra-160, SP-population specific protein ABCG2 and flk-2/flt3, but were positive for Tra-181. Altogether, these results not only consolidate previous independent reports on the phenotype and purification of in vivo human BM MSC, but also shed light on their state of differentiation and relationship with other adult stem cells.

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Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0723 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1312-1320 ◽

Cited By ~ 10

Author(s):

Ellen C. Teng ◽

Lance R. Todd ◽

Thomas J. Ribar ◽

William Lento ◽

Leah Dimascio ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Adult Stem Cells ◽

Kinase Inhibitor ◽

Embryonic Stem ◽

Mitochondrial Morphology ◽

In Vitro Proliferation ◽

Hematopoietic Stem

Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27kip1. In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27kip1. KD of Gfer results in enhanced binding of p27kip1 to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27kip1 pathway in HSCs that functions to restrict abnormal proliferation.

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Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

10.1101/126540 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yuliana Mihaylova ◽

Prasad Abnave ◽

Damian Kao ◽

Samantha Hughes ◽

Alvina Lai ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Epigenetic Regulation ◽

Cell System ◽

Stem Cell Population ◽

Loss Of Function ◽

Tumour Suppressors ◽

A Genome ◽

Regulatory Effects

AbstractCurrently, little is known about the evolution of epigenetic regulation in animal stem cells. Using the planarian stem cell system to investigate the role of the COMPASS family of MLL3/4 histone methyltransferases, we demonstrate that their role as tumour suppressors in stem cells is conserved over a large evolutionary distance in animals. This also suggested the potential conservation of a genome wide epigenetic regulation program in animal stem cells, so we assessed the regulatory effects of Mll3/4 loss of function by performing RNA-seq and ChIP-seq on the G2/M planarian stem cell population, part of which contributes to the formation of outgrowths. We find many oncogenes and tumour suppressors among the affected genes that are therefore likely candidates for mediating MLL3/4 tumour suppression function in mammals, where little is known about in vivo regulatory targets. Our work demonstrates conservation of an important epigenetic regulatory program in animals and highlights the utility of the planarian model system for studying epigenetic regulation.

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Long non-coding RNA NORAD promotes pancreatic cancer stem cell proliferation and self-renewal by blocking microRNA-202-5p-mediated ANP32E inhibition

Journal of Translational Medicine ◽

10.1186/s12967-021-03052-5 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Yu-Shui Ma ◽

Xiao-Li Yang ◽

Yu-Shan Liu ◽

Hua Ding ◽

Jian-Jun Wu ◽

...

Keyword(s):

Pancreatic Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Cell Viability ◽

Luciferase Reporter ◽

Cell Cycle Distribution ◽

Loss Of Function ◽

Self Renewal

Abstract Background Cancer stem cells (CSCs) are key regulators in the processes of tumor initiation, progression, and recurrence. The mechanism that maintains their stemness remains enigmatic, although the role of several long noncoding RNAs (lncRNAs) has been highlighted in the pancreatic cancer stem cells (PCSCs). In this study, we first established that PCSCs overexpressing lncRNA NORAD, and then investigated the effects of NORAD on the maintenance of PCSC stemness. Methods Expression of lncRNA NORAD, miR-202-5p and ANP32E in PC tissues and cell lines was quantified after RNA isolation. Dual-luciferase reporter assay, RNA pull-down and RIP assays were performed to verify the interactions among NORAD, miR-202-5p and ANP32E. We then carried out gain- and loss-of function of miR-202-5p, ANP32E and NORAD in PANC-1 cell line, followed by measurement of the aldehyde dehydrogenase activity, cell viability, apoptosis, cell cycle distribution, colony formation, self-renewal ability and tumorigenicity of PC cells. Results LncRNA NORAD and ANP32E were upregulated in PC tissues and cells, whereas the miR-202-5p level was down-regulated. LncRNA NORAD competitively bound to miR-202-5p, and promoted the expression of the miR-202-5p target gene ANP32E thereby promoting PC cell viability, proliferation, and self-renewal ability in vitro, as well as facilitating tumorigenesis of PCSCs in vivo. Conclusion Overall, lncRNA NORAD upregulates ANP32E expression by competitively binding to miR-202-5, which accelerates the proliferation and self-renewal of PCSCs.

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