Human Testis Extracellular Matrix Enhances Human Spermatogonial Stem Cell Survival In Vitro.
Ontology highlight
ABSTRACT: Impact statement
This study developed and characterized human testis extracellular matrix (htECM) and porcine testis ECM (ptECM) for testing in human spermatogonial stem cell (hSSC) culture. Results confirmed the hypothesis that ECM from the homologous species (human) and homologous tissue (testis) is optimal for maintaining hSSCs. We describe a simplified feeder-free, serum-free condition for future iterative testing to achieve the long-term goal of stable hSSC cultures. To facilitate analysis and understand the fate of hSSCs in culture, we describe a multiparameter, high-throughput, quantitative flow cytometry approach to rapidly count undifferentiated spermatogonia, differentiated spermatogonia, apoptotic spermatogonia, and proliferative spermatogonia in hSSC cultures.
SUBMITTER: Murdock MH
PROVIDER: S-EPMC6482918 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2-4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.
| S-EPMC4118148 | biostudies-literature
Project description:Spermatogonial stem cells (SSCs) possess a unique ability to recolonize the seminiferous tubules. Upon microinjection into the adluminal compartment of the seminiferous tubules, SSCs transmigrate through the blood-testis barrier (BTB) to the basal compartment of the tubule and reinitiate spermatogenesis. It was recently discovered that inhibiting retinoic acid signaling with WIN18,446 enhances SSC colonization by transiently suppressing spermatogonia differentiation, thereby promoting fertility restoration. In this study, we report that WIN18,446 increases SSC colonization by disrupting the BTB. WIN18,446 altered the expression patterns of tight junction proteins (TJPs) and disrupted the BTB in busulfan-treated mice. WIN18,446 upregulated the expression of FGF2, one of the self-renewal factors for SSCs. While WIN18,446 enhanced SSC colonization in busulfan-treated wild-type mice, it did not increase colonization levels in busulfan-treated Cldn11-deficient mice, which lack the BTB, indicating that the enhancement of SSC colonization in wild-type testes depended on the loss of the BTB. Serial transplantation analysis revealed impaired self-renewal caused by WIN18,446, indicating that WIN18,446-mediated inhibition of retinoic acid signaling impaired SSC self-renewal. Strikingly, WIN18,446 administration resulted in the death of 45% of busulfan-treated recipient mice. These findings suggest that TJP modulation is the primary mechanism behind enhanced SSC homing by WIN18,446 and raise concerns regarding the use of WIN18,446 for human SSC transplantation.
| S-EPMC10721852 | biostudies-literature
Project description:In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.
| S-EPMC5003964 | biostudies-literature
Project description:Mammalian spermatogenesis is a classic adult stems cell-dependent process, supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). However, the identification of SSCs and elucidation of their behaviors in undisturbed testis has long been a big challenge. Here, we generated a knock-in mouse model, Id4-2A-CreERT2-2A-tdTomato, which allowed us to mark Id4-expressing (Id4(+)) cells at different time points in situ and track their behaviors across distinct developmental stages during steady-state and regenerating spermatogenesis. We found that Id4(+) cells continue to produce spermatogonia, spermatocytes and sperm in mouse testis, showing they are capable of self-renewal and have differentiation potential. Consistent with these findings, ablation of Id4(+) cells in mice results in a loss of spermatogenesis. Furthermore, developmental fate mapping reveals that Id4(+) SSCs originate from neonate Id4(+) gonocytes. Therefore, our results indicate that Id4 marks spermatogonial stem cells in the mouse testis.
| S-EPMC4665196 | biostudies-literature
Project description:Myocardial infarction (MI) produces a collagen scar, altering the local microenvironment and impeding cardiac function. Cell therapy is a promising therapeutic option to replace the billions of myocytes lost following MI. Despite early successes, chronic function remains impaired and is likely a result of poor cellular retention, proliferation, and differentiation/maturation. While some efforts to deliver cells with scaffolds have attempted to address these shortcomings, they lack the natural cues required for optimal cell function. The goal of this study was to determine whether a naturally derived cardiac extracellular matrix (cECM) could enhance cardiac progenitor cell (CPC) function in vitro. CPCs were isolated via magnetic sorting of c-kit(+) cells and were grown on plates coated with either cECM or collagen I (Col). Our results show an increase in early cardiomyocyte markers on cECM compared with Col, as well as corresponding protein expression at a later time. CPCs show stronger serum-induced proliferation on cECM compared with Col, as well as increased resistance to apoptosis following serum starvation. Finally, a microfluidic adhesion assay demonstrated stronger adhesion of CPCs to cECM compared with Col. These data suggest that cECM may be optimal for CPC therapeutic delivery, as well as providing potential mechanisms to overcome the shortcomings of naked cell therapy.
| S-EPMC3488121 | biostudies-other
Project description:Mesenchymal stem cell derived extracellular matrix (MSC-ECM) is a natural biomaterial with robust bioactivity and good biocompatibility, and has been studied as a scaffold for tissue engineering. In this investigation, we tested the applicability of using decellularized human bone marrow derived MSC-ECM (hBMSC-ECM) as a culture substrate for chondrocyte expansion in vitro, as well as a scaffold for chondrocyte-based cartilage repair. hBMSC-ECM deposited by hBMSCs cultured on tissue culture plastic (TCP) was harvested, and then subjected to a decellularization process to remove hBMSCs. Compared with chondrocytes grown on TCP, chondrocytes seeded onto hBMSC-ECM exhibited significantly increased proliferation rate, and maintained better chondrocytic phenotype than TCP group. After being expanded to the same cell number and placed in high-density micromass cultures, chondrocytes from the ECM group showed better chondrogenic differentiation profile than those from the TCP group. To test cartilage formation ability, composites of hBMSC-ECM impregnated with chondrocytes were subjected to brief trypsin treatment to allow cell-mediated contraction, and folded to form 3-dimensional chondrocyte-impregnated hBMSC-ECM (Cell/ECM constructs). Upon culture in vitro in chondrogenic medium for 21 days, robust cartilage formation was observed in the Cell/ECM constructs. Similarly prepared Cell/ECM constructs were tested in vivo by subcutaneous implantation into SCID mice. Prominent cartilage formation was observed in the implanted Cell/ECM constructs 14 days post-implantation, with higher sGAG deposition compared to controls consisting of chondrocyte cell sheets. Taken together, these findings demonstrate that hBMSC-ECM is a superior culture substrate for chondrocyte expansion and a bioactive matrix potentially applicable for cartilage regeneration in vivo.Statement of significanceCurrent cell-based treatments for focal cartilage defects face challenges, including chondrocyte dedifferentiation, need for xenogenic scaffolds, and suboptimal cartilage formation. We present here a novel technique that utilizes adult stem cell-derived extracellular matrix, as a culture substrate and/or encapsulation scaffold for human adult chondrocytes, for the repair of cartilage defects. Chondrocytes cultured in stem cell-derived matrix showed higher proliferation, better chondrocytic phenotype, and improved redifferentiation ability upon in vitro culture expansion. Most importantly, 3-dimensional constructs formed from chondrocytes folded within stem cell matrix manifested excellent cartilage formation both in vitro and in vivo. These findings demonstrate the suitability of stem cell-derived extracellular matrix as a culture substrate for chondrocyte expansion as well as a candidate bioactive matrix for cartilage regeneration.
| S-EPMC5831499 | biostudies-literature
Project description:BackgroundSpermatogonial stem cells (SSCs) have the unique ability to undergo self-renewal division. However, these cells are morphologically indistinguishable from committed spermatogonia, which have limited mitotic activity. To establish a system for SSC purification, we analyzed the expression of SSC markers CD9 and epithelial cell adhesion molecule (EPCAM), both of which are also expressed on embryonic stem (ES) cells. We examined the correlation between their expression patterns and SSC activities.Methodology and principal findingsBy magnetic cell sorting, we found that EPCAM-selected mouse germ cells have limited clonogenic potential in vitro. Moreover, these cells showed stronger expression of progenitor markers than CD9-selected cells, which are significantly more enriched in SSCs. Fluorescence-activated cell sorting of CD9-selected cells indicated a significantly higher frequency of SSCs among the CD9(+)EPCAM(low/-) population than among the CD9(+)EPCAM(+) population. Overexpression of the active form of EPCAM in germline stem (GS) cell cultures did not significantly influence SSC activity, whereas EPCAM suppression by short hairpin RNA compromised GS cell proliferation and increased the concentration of SSCs, as revealed by germ cell transplantation.Conclusions/significanceThese results show that SSCs are the most concentrated in CD9(+)EPCAM(low/-) population and also suggest that EPCAM plays an important role in progenitor cell amplification in the mouse spermatogenic system. The establishment of a method to distinguish progenitor spermatogonia from SSCs will be useful for developing an improved purification strategy for SSCs from testis cells.
| S-EPMC3156235 | biostudies-literature
Project description:Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotency in vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs in in vitro cultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2-4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~?10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs during in vitro culture.
| S-EPMC3591227 | biostudies-literature
Project description:Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.
| S-EPMC3322260 | biostudies-literature
Project description:Myc plays critical roles in the self-renewal division of various stem cell types. In spermatogonial stem cells (SSCs), Myc controls SSC fate decisions because Myc overexpression induces enhanced self-renewal division, while depletion of Max, a Myc-binding partner, leads to meiotic induction. However, the mechanism by which Myc acts on SSC fate is unclear. Here we demonstrate a critical link between Myc/Mycn gene activity and glycolysis in SSC self-renewal. In SSCs, Myc/Mycn are regulated by Foxo1, whose deficiency impairs SSC self-renewal. Myc/Mycn-deficient SSCs not only undergo limited self-renewal division but also display diminished glycolytic activity. While inhibition of glycolysis decreased SSC activity, chemical stimulation of glycolysis or transfection of active Akt1 or Pdpk1 (phosphoinositide-dependent protein kinase 1 ) augmented self-renewal division, and long-term SSC cultures were derived from a nonpermissive strain that showed limited self-renewal division. These results suggested that Myc-mediated glycolysis is an important factor that increases the frequency of SSC self-renewal division.
| S-EPMC5204355 | biostudies-literature