Tissue-resident Stem Cells Research Articles

Emerging Role of Lymphocyte Antigen-6 Family of Genes in Cancer and Immune Cells.

Stem Cell Antigen-1 (Sca-1/Ly6A) was the first identified member of the Lymphocyte antigen-6 (Ly6) gene family. Sca-1 serves as a marker of cancer stem cells and tissue resident stem cells in mice. The Sca-1 gene is located on mouse chromosome 15. While a direct homolog of Sca-1 in humans is missing, human chromosome 8—the syntenic region to mouse chromosome 15—harbors several genes containing the characteristic domain known as LU domain. The function of the LU domain in human LY6 gene family is not yet defined. The LY6 gene family proteins are present on human chromosome 6, 8, 11, and 19. The most interesting of these genes are located on chromosome 8q24.3, a frequently amplified locus in human cancer. Human LY6 genes represent novel biomarkers for poor cancer prognosis and are required for cancer progression in addition to playing an important role in immune escape. Although the mechanism associated with these phenotype is not yet clear, it is timely to review the current literature in order to address the critical need for future advancements in this field. This review will summarize recent findings which describe the role of human LY6 genes—LY6D, LY6E, LY6H, LY6K, PSCA, LYPD2, SLURP1, GML, GPIHBP1, and LYNX1; and their orthologs in mice at chromosome 15.

Metabolism as an early predictor of DPSCs aging

Tissue resident adult stem cells are known to participate in tissue regeneration and repair that follows cell turnover, or injury. It has been well established that aging impedes the regeneration capabilities at the cellular level, but it is not clear if the different onset of stem cell aging between individuals can be predicted or prevented at an earlier stage. Here we studied the dental pulp stem cells (DPSCs), a population of adult stem cells that is known to participate in the repair of an injured tooth, and its properties can be affected by aging. The dental pulp from third molars of a diverse patient group were surgically extracted, generating cells that had a high percentage of mesenchymal stem cell markers CD29, CD44, CD146 and Stro1 and had the ability to differentiate into osteo/odontogenic and adipogenic lineages. Through RNA seq and qPCR analysis we identified homeobox protein, Barx1, as a marker for DPSCs. Furthermore, using high throughput transcriptomic and proteomic analysis we identified markers for DPSC populations with accelerated replicative senescence. In particular, we show that the transforming growth factor-beta (TGF-β) pathway and the cytoskeletal proteins are upregulated in rapid aging DPSCs, indicating a loss of stem cell characteristics and spontaneous initiation of terminal differentiation. Importantly, using metabolic flux analysis, we identified a metabolic signature for the rapid aging DPSCs, prior to manifestation of senescence phenotypes. This metabolic signature therefore can be used to predict the onset of replicative senescence. Hence, the present study identifies Barx1 as a DPSCs marker and dissects the first predictive metabolic signature for DPSCs aging.

Dissecting Muscle Stem Cell Aging Through Integrative Genomic Analysis

During aging, there is a progressive loss of volume and function in skeletal muscle that impacts mobility and quality of life. The repair of skeletal muscle is regulated by tissue resident stem cells called satellite cells (MuSCs), but in aging, MuSCs decrease in numbers and regenerative capacity. The transcriptional networks and epigenetic changes that confer diminished regenerative function in MuSCs as a result of natural aging are partially understood. Herein, an integrative genomics approach was utilized to profile MuSCs from young and aged animals before and after injury. Integration of these datasets revealed aging impacts multiple regulatory changes through significant differences in gene expression, metabolic flux, chromatin accessibility and patterns of transcription factor (TF) binding activities. Collectively, these datasets facilitate a deeper understanding of the regulation tissue resident stem cells utilize during aging and healing.

Quantifying proliferative and surface marker heterogeneity in colony-founding connective tissue progenitors and their progeny using time-lapse microscopy.

Connective tissue progenitors (CTPs) are defined as the heterogeneous population of tissue-resident stem and progenitor cells that are capable of proliferating and differentiating into connective tissue phenotypes. The prevalence and variation in clonal progeny of CTPs can be characterized using a colony formation assay. However, colony assays do not directly assess the characteristics of the colony-founding CTP. We performed large, field-of-view, time-lapse microscopy to manually track colonies back to the founding cells. Image processing and analysis was used to characterize the colonies and their founding cells. We found that the traditional colony-forming unit (CFU) assay underestimates the number of founding cells as colonies can be formed by more than one founding cell. After 6days in culture, colonies do not completely express CD73, CD90, and CD105. Heterogeneity in colony cells was characterized by two cell populations, proliferative and spread cells. Regression modelling of duration of lag phase and doubling time by cell marker suggests the presence of CD90 and CD105 in CTP subpopulations with different proliferative capabilities. From mathematical modelling of clonal colonies, we quantitatively characterized proliferation, migration, and cell marker expression rates to identify desirable clones for selection. Direct assessment of colony formation parameters led to more accurate assessment of CFU heterogeneity. Furthermore, these parameters can be used to quantify the diversity and hierarchy of stem and progenitor cells from a cell source or tissue for tissue engineering applications.

Muscle Satellite Cell Cross-Talk with a Vascular Niche Maintains Quiescence via VEGF and Notch Signaling

Skeletal muscle is a complex tissue containing tissue resident muscle stem cells (satellite cells) (MuSCs) important for postnatal muscle growth and regeneration. Quantitative analysis of the biological functionof MuSCs and the molecular pathways responsible for a potential juxtavascular niche for MuSCs is currently lacking. We utilized fluorescent reporter mice and muscle tissue clearing to investigate the proximity of MuSCs to capillaries in 3 dimensions. We show that MuSCs express abundant VEGFA, which recruits endothelial cells (ECs) invitro, whereas blocking VEGFA using both a vascular endothelial growth factor (VEGF) inhibitor and MuSC-specific VEGFA gene deletion reduces the proximity of MuSCs to capillaries. Importantly, this proximity to the blood vessels was associated with MuSC self-renewal in which the EC-derived Notch ligand Dll4 induces quiescence in MuSCs. We hypothesize that MuSCs recruit capillary ECs via VEGFA, and in return, ECs maintain MuSC quiescence though Dll4.

Initiation, Expansion, and Cryopreservation of Human Primary Tissue‐Derived Normal and Diseased Organoids in Embedded Three‐Dimensional Culture

Organoids are primary patient-derived micro tissues grown within a three-dimensional extracellular matrix that better represents in vivo physiology and genetic diversity than existing two-dimensional cell lines. Organoids rely on the self-renewal and differentiation of tissue-resident stem cells that expand in culture and self-organize into complex three-dimensional structures. Depending on the tissue, organoids typically lack stromal, vascular, neural, and immune cells but otherwise can contain cells from all the respective tissue-specific cell lineages found in vivo. Established organoids can be initiated from cryopreserved material, cultured using largely traditional cell culture techniques and equipment, and then expanded and cryopreserved for future use. Organoid models have been developed from a variety of diseased and normal tissues including small intestine, colon, mammary, esophagus, lung, prostate, and pancreas. © 2018 by John Wiley & Sons, Inc.

Hoxc-Dependent Mesenchymal Niche Heterogeneity Drives Regional Hair Follicle Regeneration

Mesenchymal niche cells instruct activity of tissue-resident stem and progenitor cell populations. Epithelial stem cells in hair follicles (HFs) have region-specific activity, which may arise from intrinsic cellular heterogeneity within mesenchymal dermal papilla (DP) cells. Here we show that expression of Hoxc genes is sufficient to reprogram mesenchymal DP cells and alter the regenerative potential of epithelial stem cells. Hoxc gene expression in adult skin dermis closely correlates with regional HF regeneration patterns. Disrupting the region-specific expression patterns of Hoxc genes, by either decreasing their epigenetic repression via Bmi1 loss or inducing ectopic interactions of the Hoxc locus with an active epigenetic region, leads to precocious HF regeneration. We further show that a single Hoxc gene is sufficient to activate dormant DP niches and promote regional HF regeneration through canonical Wnt signaling. Altogether, these results reveal that Hoxc genes bestow mesenchymal niches with tissue-level heterogeneity and plasticity.

Signaling in the stem cell niche: regulating cell fate, function and plasticity.

Stem cells have the ability to self-renew and differentiate along multiple lineages, driving tissue homeostasis and regeneration. Paradigms of unidirectional, hierarchical differentiation trajectories observed in embryonic and hematopoietic stem cells have traditionally been applied to tissue-resident stem cells. However, accumulating evidence implicates stemness as a bidirectional, dynamic state that is largely governed by the niche, which facilitates plasticity and adaptability to changing conditions. In this Review, we discuss mechanisms of cell fate regulation through niche-derived cues, with a particular focus on epithelial stem cells of the mammalian skin, intestine and lung. We discuss a spectrum of niche-derived biochemical, mechanical and architectural inputs that define stem cell states during morphogenesis, homeostasis and regeneration, and highlight how these diverse inputs influence stem cell plasticity.

Origin of tendon stem cells in situ

Adult stem cells are surveillance repositories capable of supplying a renewable source of progenitors for tissue repair and regeneration to maintain tissue homeostasis throughout life. Many tissue-resident stem cells have been identified in situ, which lays the foundation for studying them in their native microenvironment, i.e. the niche. Within the musculoskeletal system, muscle stem cells have been unequivocally identified in the mouse, which have led to considerable advances in understanding their role in muscle homeostasis and regeneration. On the other hand, for bone and tendon progenitor cells, mesenchymal stem cells have been used as the main in vitro cell model as they can differentiate into osteogenic, chondrogenic and tenogenic fates. Despite considerable efforts and employment of modern tools, the in vivo origins of bone and tendon stem cells remain debated. Tendon regeneration via stem cells is understudied and deserves attention as tendon damage is noted for a bleak, time-consuming recovery and the repaired tendon seldom regains the structural integrity and strength of the native, uninjured state. Here we review the past efforts and recent studies toward defining adult tendon stem cells and understanding tendon regeneration instead of tendon development. The focus is on adult tendon resident cells in situ and the uncertainty of their roles in regeneration. A systematic literature search using the Pubmed search engine was conducted encompassing the seminal papers in the tendon field. Investigation of tendon stem cells in situ is in its infancy mainly due to lack of necessary tools and standardized injury model. We propose a concerted effort toward establishing a comprehensive cell atlas of the tendon, making genetic tools and choosing a reliable injury model for coordinated studies among different laboratories. Increasing our basic understanding should aid future therapeutic innovations to shorten and enhance the tendon repair/regeneration process.

The Role of Microenvironment in Preserving the Potency of Adult Porcine Pulmonary Valve Stem Cells In Vitro.

Background and ObjectiveThe potency of tissue resident stem cells is regulated primarily by inputs from the local microenvironment. Isolation of stem cells through enzymatic digestion of tissue may affect epigenetic regulation of cell fate and performance. Here we employ a non-enzymatic method to harvest and investigate tissue resident stem cells from the adult porcine pulmonary valve.Methods and ResultsThe presence of c-Kit+ stem cells within the valve tissue was confirmed by immunohistochemistry. An in vitro culture of minced valve leaflets was developed under the standard conditions (37°C with 5% CO2). The viability of the cellular outgrowths was evaluated over the subsequent 12 weeks. Under this culture condition, we identified a population of non-adherent c-Kit+ cells and multiple cellular structures mimicking the phenotype of embryonic stem cells at different stages of development. Formation of multinucleated cells through cell fusion provided an active niche area for homing and interaction of the non-adherent c-Kit+ cells. Expression of pluripotency markers Oct-4 and Nanog was detected in the newly formed multinucleated cells but not in mature colonies. Partial cell fusion was shown by fluorescent live-cell tracking, which confirmed intercellular molecular exchange between donor and recipient cells, resulting in altered cytoplasmic protein expression by the recipient cell.ConclusionsThese results suggest a role for the microenvironment in decrypting the potential of the valve somatic stem cells in vitro. In addition, our data provide evidence for cell fusion, which may play a critical role in reversing somatic cell fate and spontaneous cellular reprogramming.

Mesenchymal Stem Cells: Miraculous Healers or Dormant Killers?

Mesenchymal Stem Cells (MSCs) are a heterogeneous population of fibroblast-like cells which maintain self-renewability and pluripotency to differentiate into mesodermal cell lineages. The use of MSCs in clinical settings began with high enthusiasm and the number of MSC-based clinical trials has been rising ever since. However; the very unique characteristics of MSCs that made them suitable to for therapeutic use, might give rise to unwanted outcomes, including tumor formation and progression. In this paper, we present a model of carcinogenesis initiated by MSCs, which chains together the tissue organization field theory, the stem cell theory, and the inflammation-cancer chain. We believe that some tissue resident stem cells could be leaked cells from bone marrow MSC pool to various injured tissue, which consequently transform and integrate in the host tissue. If the injury persists or chronic inflammation develops, as a consequence of recurring exposure to growth factors, cytokines, etc. the newly formed tissue from MSCs, which still has conserved their mesenchymal and stemness features, go through rapid population expansion, and nullify their tumor suppressor genes, and hence give rise to neoplastic cell (carcinomas, sarcomas, and carcino-sarcomas). Considering the probability of this hypothesis being true, the clinical and therapeutic use of MSCs should be with caution, and the recipients' long term follow-up seems to be insightful.

Muscle stem cell intramuscular delivery within hyaluronan methylcellulose improves engraftment efficiency and dispersion

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. We demonstrate that increases in donor-derived fibers when using HAMC are attributed to increased MuSC proliferation via a CD44-independent mechanism, preventing injected cell active clearance, and supporting in vivo expansion by delaying differentiation. Furthermore, we observed a significant improvement in donor fiber dispersion when MuSCs were delivered in HAMC. Our study results suggest that HAMC is a promising muscle stem cell delivery vehicle.

Stem cell properties of human clonal salivary gland stem cells are enhanced by three-dimensional priming culture in nanofibrous microwells

BackgroundThree-dimensional (3D) cultures recapitulate the microenvironment of tissue-resident stem cells and enable them to modulate their properties. We determined whether salivary gland-resident stem cells (SGSCs) are primed by a 3D spheroid culture prior to treating irradiation-induced salivary hypofunction using in-vitro coculture and in-vivo transplant models.Methods3D spheroid-derived SGSCs (SGSCs3D) were obtained from 3D culture in microwells consisting of a nanofiber bottom and cell-repellent hydrogel walls, and were examined for salivary stem or epithelial gene/protein expression, differentiation potential, and paracrine secretory function compared with monolayer-cultured SGSCs (SGSCs2D) in vitro and in vivo.ResultsSGSCs3D expressed increased salivary stem cell markers (LGR5 and THY1) and pluripotency markers (POU5F1 and NANOG) compared with SGSCs2D. Also, SGSCs3D exhibited enhanced potential to differentiate into salivary epithelial cells upon differentiation induction and increased paracrine secretion as compared to SGSCs2D. Wnt signaling was activated by 3D spheroid formation in the microwells and suppression of the Wnt/β-catenin pathway led to reduced stemness of SGSCs3D. Enhanced radioprotective properties of SGSCs3D against radiation-induced salivary hypofunction was confirmed by an organotypic 3D coculture and in-vivo transplantation experiments.ConclusionThe 3D spheroid culture of SGSCs in nanofibrous microwells promotes stem cell properties via activation of Wnt signaling. This may contribute to SGSC priming prior to regenerative therapy to restore salivary hypofunction after radiotherapy.

Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila.

Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells’ environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly’s survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

Therapeutic effects of a recombinant human collagen peptide bioscaffold with human adipose-derived stem cells on impaired wound healing after radiotherapy.

Chronic changes following radiotherapy include alterations in tissue-resident stem cells and vasculatures, which can lead to impaired wound healing. In this study, novel recombinant human collagen peptide (rhCP) scaffolds were evaluated as a biomaterial carrier for cellular regenerative therapy. Human adipose-derived stem cells (hASCs) were successfully cultured on rhCP scaffolds. By hASC culture on rhCP, microarray assay indicated that expression of genes related to cell proliferation and extracellular matrix production was upregulated. Pathway analyses revealed that signaling pathways related to inflammatory suppression and cell growth promotion were activated as well as signaling pathways consistent with some growth factors including vascular endothelial growth factor, hepatocyte growth factor, and transforming growth factor beta, although gene expression of these growth factors was not upregulated. These findings suggest the rhCP scaffold showed similar biological actions to cytokines regulating cell growth and immunity. In subsequent impaired wound healing experiments using a locally irradiated (20 Gray) mouse, wound treatment with rhCP sponges combined with cultured hASCs and human umbilical vein endothelial cells accelerated wound closure compared with wounds treated with rhCP with hASCs alone, rhCP only, and control (dressing alone), with better healing observed according to this order. These results indicating the therapeutic value of rhCP scaffolds as a topical biomaterial dressing and a biocarrier of stem cells and vascular endothelial cells for regenerating therapies. The combination of rhCP and functional cells was suggested to be a potential tool for revitalizing stem cell-depleted conditions such as radiation tissue damage.

Anterior cruciate ligament-derived mesenchymal stromal cells have a propensity to differentiate into the ligament lineage.

IntroductionThe anterior cruciate ligament (ACL) consists of various components, such as collagen, elastin fibres, and fibroblasts. Because ACL has a poor regenerative ability, ACL reconstruction need require the use of autologous tendons. In recent years, tissue-resident stem cells have been studied to promote ACL regeneration as an alternatively method. However, the existence of stem cells in ligaments has not been clearly defined. Here, we prospectively isolated stem cells from ACLs and characterized their properties.MethodsACLs from 11 donors and bone marrows (BM) from 8 donors were obtained with total knee arthroplasty. We used flow cytometry to screen the cell surface markers on ACL cells. Frozen sections were prepared from patient ACL tissues and stained with specific antibodies. Cultured ACL-derived and BM-derived cells at passage 3 were differentiated into adipocytes, osteoblasts and tendon/ligament cells.ResultsACL-derived mesenchymal stem/stromal cells (ACL-MSCs) expressed high levels of CD73 and CD90. Immunohistochemical analyses revealed that ACL-MSCs were located on the inner surface of ACL sinusoids. Furthermore, the expression of cell surface antigens was clearly different between ACL-MSCs and bone marrow (BM)-derived MSCs (BM-MSCs) at the time of isolation, but the two cell populations became indistinguishable after long-term culture. Interestingly, ACL-MSCs are markedly different from BM-MSCs in their differentiation ability and have a high propensity to differentiate into ligament-committed cells.ConclusionsOur findings suggest that ACL-MSCs express CD90 and CD73 markers, and their differentiation capacity is maintained even through culture. The cell population having tissue-specific properties is an important research target for investigating the ligament therapies.

Immunosuppression by Intestinal Stromal Cells.

This chapter summarizes evidence that intestinal myofibroblasts, also called intestinal stromal cells, are derived in the adult from tissue mesenchymal stem cells under homeostasis and may be replenished by bone marrow mesenchymal stromal (stem) cells that are recruited after severe intestinal injury. A comparison of mechanism of immunosuppression or tolerance by adult intestinal stromal cells (myofibroblasts) is almost identical with those reported for mesenchymal stem cells of bone marrow origin. The list of suppression mechanisms includes PD-L1 and PD-L2/PD-1 immune checkpoint pathways, soluble mediator secretion, toll-like receptor-mediated tolerance, and augmentation of Treg cells. Further, both mesenchymal stem cells and intestinal stromal cells express an almost identical repertoire of CD molecules. Lastly, others have reported that isolate intestinal stromal cells are capable of differentiating into bone and less well into chondrocyte, but not into adipocytes, a finding that we have confirmed. These findings suggest that intestinal stromal cells (myofibroblasts) are partially differentiated adult, tissue-resident stem cells which are capable of exerting immune tolerance in the intestine. Their role in repair of inflammatory bowel disease and immune suppression in colorectal cancer needs further investigation.

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.

Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory Tcells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs.

Stem cell dynamics in muscle regeneration: Insights from live imaging in different animal models.

In recent years, live imaging has been adopted to study stem cells in their native environment at cellular resolution. In the skeletal muscle field, this has led to visualising the initial events of muscle repair in mouse, and the entire regenerative response in zebrafish. Here, we review recent discoveries in this field obtained from live imaging studies. Tracking of tissue resident stem cells, the satellite cells, following injury has captured the morphogenetic dynamics of stem/progenitor cells as they facilitate repair. Asymmetric satellite cell division generated a clonogenic progenitor pool, providing in vivo validation for this mechanism. Furthermore, there is an emerging role of stem/progenitor cell guidance at the injury site by cellular protrusions. This review concludes that live imaging is a critical tool for discovering the distinct processes that occur during regeneration, emphasising the importance of imaging in diverse animal models to capture the entire scope of stem cell functions. Also see the Video Abstract. Link to: https://youtube/tgUHSBD1N0g.