Optimization of decellularized human placental macroporous scaffolds for spermatogonial stem cells homing.
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ABSTRACT:
SUBMITTER: Asgari F
PROVIDER: S-EPMC8065005 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm(2), 10 wt%, 6330±1160 µm(2), 20 wt%, 7600±1550 µm(2)) compared with controls (0 wt%, 3150±220 µm(2)). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.
| S-EPMC3492243 | biostudies-literature
Project description:Generating new kidneys using tissue engineering technologies is an innovative strategy for overcoming the shortage of donor organs for transplantation. Here we report how to efficiently engineer the kidney vasculature of decellularized rat kidney scaffolds by using human induced pluripotent stem cell (hiPSCs)-derived endothelial cells (hiPSC-ECs). In vitro, hiPSC-ECs responded to flow stress by acquiring an alignment orientation, and attached to and proliferated on the acellular kidney sections, maintaining their phenotype. The hiPSC-ECs were able to self-organize into chimeric kidney organoids to form vessel-like structures. Ex vivo infusion of hiPSC-ECs through the renal artery and vein of acellular kidneys resulted in the uniform distribution of the cells in all the vasculature compartments, from glomerular capillaries to peritubular capillaries and small vessels. Ultrastructural analysis of repopulated scaffolds through transmission and scanning electron microscopy demonstrated the presence of continuously distributed cells along the vessel wall, which was also confirmed by 3D reconstruction of z-stack images showing the continuity of endothelial cell coverage inside the vessels. Notably, the detection of fenestrae in the endothelium of glomerular capillaries but not in the vascular capillaries was clear evidence of site-specific endothelial cell specialisation.
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Project description:Study questionCan human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia?Summary answerGerm cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures.What is known alreadyThe development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures.Study design, size, durationTesticular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures.Participants/materials, setting, methodsImmunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4).Main results and the role of chanceUTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected.Limitations, reasons for cautionDifferent groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences.Wider implications of the findingsThe ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy.Study funding/competing interestsThe experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests.Trial registration numberNot applicable.
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Project description:Permanent sensorineural hearing loss is a major medical problem and is due to the loss of hair cells and subsequently spiral ganglion neurons in the cochlea. Since these cells lack the capacity of renewal in mammals, their regeneration would be an optimal solution to reverse hearing loss. In other tissues, decellularized extracellular matrix (ECM) has been used as a mechanical and biochemical scaffold for the induction of stem and other cells toward a target tissue phenotype. Such induced cells have been used for tissue and organ transplants in preclinical animal and human clinical applications. This paper reports for the first time the decellularization of the cochlea and identification of remaining laminin and collagen type IV as a first step in preparing an ECM scaffold for directing stem cells toward an auditory lineage. Fresh ear tissues were removed from euthanized mice, a rat and a human and processed for decellularization using two different detergent extraction methods. Cochleas were imaged with scanning thin-sheet laser imaging microscopy (sTSLIM) and brightfield microscopy. Detergent treatment of fresh tissue removed all cells as evidenced by lack of H&E and DNA staining of the membranous labyrinth while preserving components of the ECM. The organ of Corti was completely removed, as were spiral ganglion neurons, which appeared as hollow sheaths and tubes of basal lamina (BL) material. Cells of the stria vascularis were removed and its only vestige left was its laterally linking network of capillary BL that appeared to "float" in the endolymphatic space. Laminin and type IV collagen were detected in the ECM after decellularization and were localized in vascular, neural and epithelial BL. Further work is necessary to attempt to seed neural and other stem cells into the decellularized ECM to hopefully induce differentiation and subsequent in vivo engraftment into damaged cochleas.
| S-EPMC3540273 | biostudies-literature
Project description:Decellularized tissues have become a common regenerative medicine platform with multiple materials being researched in academic laboratories, tested in animal studies, and used clinically. Ideally, when a tissue is decellularized the native cell niche is maintained with many of the structural and biochemical cues that naturally interact with the cells of that particular tissue. This makes decellularized tissue materials an excellent platform for providing cells with the signals needed to initiate and maintain differentiation into tissue-specific lineages. The extracellular matrix (ECM) that remains after the decellularization process contains the components of a tissue specific microenvironment that is not possible to create synthetically. The ECM of each tissue has a different composition and structure and therefore has unique properties and potential for affecting cell behavior. This review describes the common methods for preparing decellularized tissue materials and the effects that decellularized materials from different tissues have on cell phenotype.
| S-EPMC4979580 | biostudies-literature
Project description:Identification of appropriate donor cell types is important for lung cell therapy and for lung regeneration. Previous studies have indicated that mesenchymal stromal cells derived from human bone marrow (hBM-MSCs) and from human adipose tissue (hAT-MSCs) may have the ability to trans-differentiate into lung epithelial cells. However, these data remain controversial. Herein, the ability of hBM-MSCs and hAT-MSCs to repopulate acellular rodent lung tissue was evaluated. hBM-MSCs and hAT-MSCs were isolated from bone marrow aspirate and lipoaspirate, respectively. Rat lungs were decellularized with CHAPS detergent, followed by seeding the matrix with hBM-MSCs and hAT-MSCs. Under appropriate culture conditions, both human MSC populations attached to and proliferated within the lung tissue scaffold. In addition, cells were capable of type 2 pneumocyte differentiation, as assessed by marker expression of surfactant protein C (pro-SPC) at the protein and the RNA level, and by the presence of lamellar bodies by transmission electron microscopy. Additionally, hAT-MSCs contributed to Clara-like cells that lined the airways in the lung scaffolds, whereas the hBM-MSCs did not. We also tested the differentiation potential of MSCs on different extracellular matrix components in vitro, and found that protein substrate influences MSC epithelial differentiation. Together our data show the capacity for human MSCs to differentiate toward lung epithelial phenotypes, and the possibility of using these cells for lung cell therapies and tissue engineering.
| S-EPMC4029048 | biostudies-literature
Project description:Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.
| S-EPMC10226497 | biostudies-literature
Project description:Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4-contaminated human testicular cell suspensions was performed to isolate EpCAM+/HLA-ABC-/CD49e- (putative spermatogonia) and EpCAM-/HLA-ABC+/CD49e+ (putative MOLT-4) cell fractions. The EpCAM+/HLA-ABC-/CD49e- fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to-nude mouse xenotransplantation. The EpCAM-/HLA-ABC+/CD49e+ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.
| S-EPMC3613920 | biostudies-literature
Project description:ObjectivesThe aim of this study was to evaluate the level of odontogenic differentiation of dental pulp stem cells (DPSCs) on hydrogel scaffolds derived from bone extracellular matrix (bECM) in comparison to those seeded on collagen I (Col-I), one of the main components of dental pulp ECM.MethodsDPSCs isolated from human third molars were characterized for surface marker expression and odontogenic potential prior to seeding into bECM or Col-I hydrogel scaffolds. The cells were then seeded onto bECM and Col-I hydrogel scaffolds and cultured under basal conditions or with odontogenic and growth factor (GF) supplements. DPSCs cultivated on tissue culture polystyrene (TCPS) with and without supplements were used as controls. Gene expression of dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE) was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and mineral deposition was observed by Von Kossa staining.ResultsWhen DPSCs were cultured on bECM hydrogels, the mRNA expression levels of DSPP, DMP-1 and MEPE genes were significantly upregulated with respect to those cultured on Col-I scaffolds or TCPS in the absence of extra odontogenic inducers. In addition, more mineral deposition was observed on bECM hydrogel scaffolds as demonstrated by Von Kossa staining. Moreover, DSPP, DMP-1 and MEPE mRNA expressions of DPSCs cultured on bECM hydrogels were further upregulated by the addition of GFs or osteo/odontogenic medium compared to Col-I treated cells in the same culture conditions.SignificanceThese results demonstrate the potential of the bECM hydrogel scaffolds to stimulate odontogenic differentiation of DPSCs.
| S-EPMC4755593 | biostudies-literature
Project description:OBJECTIVES:This study is designed to generate and propagate human spermatogonial stem cells (SSCs) derived from human pluripotent stem cells (hPSCs). METHODS:hPSCs were differentiated into SSC-like cells (SSCLCs) by a three-step strategy. The biological characteristics of SSCLCs were detected by immunostaining with antibodies against SSC markers. The ability of self-renewal was measured by propagating for a long time and still maintaining SSCs morphological property. The differentiation potential of SSCLCs was determined by the generation of spermatocytes and haploid cells, which were identified by immunostaining and flow cytometry. The transcriptome analysis of SSCLCs was performed by RNA sequencing. The biological function of SSCLCs was assessed by xeno-transplantation into busulfan-treated mouse testes. RESULTS:SSCLCs were efficiently generated by a 3-step strategy. The SSCLCs displayed a grape-like morphology and expressed SSC markers. Moreover, SSCLCs could be propagated for approximately 4?months and still maintained their morphological properties. Furthermore, SSCLCs could differentiate into spermatocytes and haploid cells. In addition, SSCLCs displayed a similar gene expression pattern as human GPR125+ spermatogonia derived from human testicular tissues. And more, SSCLCs could survive and home at the base membrane of seminiferous tubules. CONCLUSION:SSCLCs were successfully derived from hPSCs and propagated for a long time. The SSCLCs resembled their counterpart human GPR125+ spermatogonia, as evidenced by the grape-like morphology, transcriptome, homing, and functional characteristics. Therefore, hPSC-derived SSCLCs may provide a reliable cell source for studying human SSCs biological properties, disease modeling, and drug toxicity screening.
| S-EPMC7509941 | biostudies-literature