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BACKGROUND: Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages. In humans, their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs. Studies suggested that mesenchymal stem cells (MSCs), necessary for repair and regeneration via transplantation, require doses ranging from 10 to 400 million cells. Furthermore, the limited expansion of MSCs restricts their therapeutic application. AIM: To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols. METHODS: Human umbilical cord (hUC) tissue derived MSCs were obtained and re-cultured. These cultured cells were subjected to the following evaluation procedures: Immunophenotyping, immunocytochemical staining, trilineage differentiation, population doubling time and number, gene expression markers for proliferation, cell cycle progression, senescence-associated ß-galactosidase assay, human telomerase reverse transcriptase (hTERT) expression, mycoplasma, cytomegalovirus and endotoxin detection. RESULTS: Analysis of pluripotent gene markers Oct4, Sox2, and Nanog in recultured hUC-MSC revealed no significant differences. The immunophenotypic markers CD90, CD73, CD105, CD44, vimentin, CD29, Stro-1, and Lin28 were positively expressed by these recultured expanded MSCs, and were found negative for CD34, CD11b, CD19, CD45, and HLA-DR. The recultured hUC-MSC population continued to expand through passage 15. Proliferative gene expression of Pax6, BMP2, and TGFb1 showed no significant variation between recultured hUC-MSC groups. Nevertheless, a significant increase (P < 0.001) in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs. Cellular senescence markers (hTERT expression and ß-galactosidase activity) did not show any negative effect on recultured hUC-MSCs. Additionally, quality control assessments consistently confirmed the absence of mycoplasma, cytomegalovirus, and endotoxin contamination. CONCLUSION: This study proposes the development of a novel protocol for efficiently expanding stem cell population. This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.
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BACKGROUND: Zinc (Zn) is the second most abundant trace element after Fe, present in the human body. It is frequently reported in association with cell growth and proliferation, and its deficiency is considered to be a major disease contributing factor. AIM: To determine the effect of Zn on in vitro growth and proliferation of human umbilical cord (hUC)-derived mesenchymal stem cells (MSCs). METHODS: hUC-MSCs were isolated from human umbilical cord tissue and characterized based on immunocytochemistry, immunophenotyping, and tri-lineage differentiation. The impact of Zn on cytotoxicity and proliferation was determined by MTT and Alamar blue assay. To determine the effect of Zn on population doubling time (PDT), hUC-MSCs were cultured in media with and without Zn for several passages. An in vitro scratch assay was performed to analyze the effect of Zn on the wound healing and migration capability of hUC-MSCs. A cell adhesion assay was used to test the surface adhesiveness of hUC-MSCs. Transcriptional analysis of genes involved in the cell cycle, proliferation, migration, and self-renewal of hUC-MSCs was performed by quantitative real-time polymerase chain reaction. The protein expression of Lin28, a pluripotency marker, was analyzed by immunocytochemistry. RESULTS: Zn at lower concentrations enhanced the rate of proliferation but at higher concentrations (> 100 µM), showed concentration dependent cytotoxicity in hUC-MSCs. hUC-MSCs treated with Zn exhibited a significantly greater healing and migration rate compared to untreated cells. Zn also increased the cell adhesion rate, and colony forming efficiency (CFE). In addition, Zn upregulated the expression of genes involved in the cell cycle (CDC20, CDK1, CCNA2, CDCA2), proliferation (transforming growth factor ß1, GDF5, hypoxia-inducible factor 1α), migration (CXCR4, VCAM1, VEGF-A), and self-renewal (OCT4, SOX2, NANOG) of hUC-MSCs. Expression of Lin28 protein was significantly increased in cells treated with Zn. CONCLUSION: Our findings suggest that zinc enhances the proliferation rate of hUC-MSCs decreasing the PDT, and maintaining the CFE. Zn also enhances the cell adhesion, migration, and self-renewal of hUC-MSCs. These results highlight the essential role of Zn in cell growth and development.
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Long-term in vitro culture of human mesenchymal stem cells (MSCs) leads to cell lifespan shortening and growth stagnation due to cell senescence. Here, using sequencing data generated in the public domain, we have established a specific regulatory network of "transcription factor (TF)-microRNA (miRNA)-Target" to provide key molecules for evaluating the passage-dependent replicative senescence of mesenchymal stem cells for the quality control and status evaluation of mesenchymal stem cells prepared by different procedures. Short time-series expression miner (STEM) analysis was performed on the RNA-seq and miRNA-seq databases of mesenchymal stem cells from various passages to reveal the dynamic passage-related changes of miRNAs and mRNAs. Potential miRNA targets were predicted using seven miRNA target prediction databases, including TargetScan, miRTarBase, miRDB, miRWalk, RNA22, RNAinter, and TargetMiner. Then use the TransmiR v2.0 database to obtain experimental-supported transcription factor for regulating the selected miRNA. More than ten sequencing data related to mesenchymal stem cells or mesenchymal stem cells reprogramming were used to validate key miRNAs and mRNAs. And gene set variation analysis (GSVA) was performed to calculate the passage-dependent signature. The results showed that during the passage of mesenchymal stem cells, a total of 29 miRNAs were gradually downregulated and 210 mRNA were gradually upregulated. Enrichment analysis showed that the 29 miRNAs acted as multipotent regulatory factors of stem cells and participated in a variety of signaling pathways, including TGF-beta, HIPPO and oxygen related pathways. 210 mRNAs were involved in cell senescence. According to the target prediction results, the targets of these key miRNAs and mRNAs intersect to form a regulatory network of "TF-miRNA-Target" related to replicative senescence of cultured mesenchymal stem cells, across 35 transcription factor, 7 miRNAs (has-mir-454-3p, has-mir-196b-5p, has-mir-130b-5p, has-mir-1271-5p, has-let-7i-5p, has-let-7a-5p, and has-let-7b-5p) and 7 predicted targets (PRUNE2, DIO2, CPA4, PRKAA2, DMD, DDAH1, and GATA6). This network was further validated by analyzing datasets from a variety of mesenchymal stem cells subculture and lineage reprogramming studies, as well as qPCR analysis of early passages mesenchymal stem cells versus mesenchymal stem cells with senescence morphologies (SA-ß-Gal+). The "TF-miRNA-Target" regulatory network constructed in this study reveals the functional mechanism of miRNAs in promoting the senescence of MSCs during in vitro expansion and provides indicators for monitoring the quality of functional mesenchymal stem cells during the preparation and clinical application.
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Hematopoietic stem cell (HSC) transplantation remains the most effective therapy for hematologic and lymphoid disorders. However, as the primary therapeutic cells, the source of HSCs has been limited due to the scarcity of matched donors and difficulties in ex vivo expansion. Here, we described a facile method to attempt the expansion of HSCs in vitro through a porous alginate hydrogel-based 3D culture system. We used gelatin powders as the porogen to create submillimeter-scaled pores in alginate gel bulk while pre-embedding naïve HSCs in the gel phase. The results indicated that this porous hydrogel system performed significantly better than those cultured via conventional suspension or encapsulation in non-porous alginate hydrogels in maintaining the phenotype and renewability of HSCs. Only the porous hydrogel system achieved a two-fold growth of CD34+ cells within seven days of culture, while the number of CD34+ cells in the suspension system and nonporous hydrogel showed different degrees of attenuation. The expansion efficiency of the porous hydrogel for CD34+CD38- cells was more than 2.2 times that of the other two systems. Mechanistic study via biophysical analysis revealed that the porous alginate system was competent to reduce the electron capture caused by biomaterials, decrease cellular oxygen stress, avoid oxidative protection, thus maintaining the cellular phenotype of the CD34+ cells. The transcriptomic analysis further suggested that the porous alginate system also upregulated the TNF signaling pathway and activated the NF-κB signaling pathway to promote the CD34+ cells' survival and maintain cellular homeostasis so that renewability was substantially favoured. STATEMENT OF SIGNIFICANCE: ⢠The reported porous hydrogel system performs significantly better in terms of maintaining the phenotype and renewability of HSCs than those cultured via conventional suspension or encapsulation in non-porous alginate hydrogel. ⢠The reported porous alginate system is competent to reduce the electron capture caused by biomaterials, decrease cellular oxygen stress, avoid oxidative protection, and therefore maintain the cellular phenotype of the CD34+ cells. ⢠The reported porous alginate system can also upregulate the TNF signaling pathway and activate the NF-κB signaling pathway to promote the CD34+ cells' survival and maintain cellular homeostasis so that the renewability is substantially favored..
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Células Madre Hematopoyéticas , FN-kappa B , Células Cultivadas , FN-kappa B/metabolismo , Hidrogeles/farmacología , Hidrogeles/metabolismo , Materiales Biocompatibles/metabolismo , Alginatos/farmacología , Oxígeno/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular , Sangre FetalRESUMEN
Multiple clinical trials exploring the potential of adoptive natural killer (NK) cell therapy for cancer have employed ex vivo expansion using feeder cells to obtain large numbers of NK cells. We have previously utilized the rhesus macaque model to clonally track the NK cell progeny of barcode-transduced CD34+ stem and progenitor cells after transplant. In this study, NK cells from barcoded rhesus macaques were used to study the changes in NK cell clonal patterns that occurred during ex vivo expansion using culture protocols similar to those employed in clinical preparation of human NK cells including irradiated lymphoblastoid cell line (LCL) feeder cells or K562 cells expressing 4-1BBL and membrane-bound interleukin-21 (IL-21). NK expansion cultures resulted in the proliferation of clonally diverse NK cells, which, at day 14 harvest, contained greater than 50% of the starting barcode repertoire. Diversity as measured by Shannon index was maintained after culture. With both LCL and K562 feeders, proliferation of long-lived putative memory-like NK cell clones was observed, with these clones continuing to constitute a mean of 31% of the total repertoire of expanded cells. These experiments provide insight into the clonal makeup of expanded NK cell clinical products.
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Extracellular matrices (ECM) present in our tissues play a significant role in maintaining tissue homeostasis through various physical and chemical cues such as topology, stiffness, and secretion of biochemicals. They are known to influence the behavior of resident stem cells. It is also known that ECM type and coating density on cell culture plates strongly influence in vitro cellular behavior. However, the influence of ECM protein coating on long-term mesenchymal stem cell expansion has not been studied yet. To address this gap, we cultured bone-marrow derived hMSCs for multiple passages on the tissue culture plastic plates coated with 25 µg/ml of various ECM proteins. We found that cells on plates coated with ECM proteins had much higher proliferation compared to the regular tissue culture plates. Further, gelatin-coated plates helped the cells to grow faster compared to collagen, fibronectin, and laminin coated plates. Additionally, the use of gelatin showed less size heterogeneity among the cells when expanded from passages 3 to 9 (P3 to P9). Gelatin also helped in maintaining cellular stiffness which was not observed across other ECM proteins. In summary, in this research, we have shown that gelatin which is the least expensive compared to other ECM proteins, provides a better platform for mesenchymal stem cell expansion.
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Proteínas de la Matriz Extracelular , Células Madre Mesenquimatosas , Proteínas de la Matriz Extracelular/metabolismo , Gelatina/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Diferenciación CelularRESUMEN
Mesenchymal-derived stromal or progenitor cells, commonly called "MSCs," have attracted significant clinical interest for their remarkable abilities to promote tissue regeneration and reduce inflammation. Recent studies have shown that MSCs' therapeutic effects, originally attributed to the cells' direct differentiation capacity into the tissue of interest, are largely driven by the biomolecules the cells secrete, including cytokines, chemokines, growth factors, and extracellular vesicles containing miRNA. This secretome coordinates upregulation of endogenous repair and immunomodulation in the local microenvironment through crosstalk of MSCs with host tissue cells. Therapeutic applications for MSCs and their secretome-derived products often involve in vitro monolayer expansion. However, consecutive passaging of MSCs significantly alters their therapeutic potential, inducing a broad shift from a pro-regenerative to a pro-inflammatory phenotype. A consistent by-product of in vitro expansion of MSCs is the onset of replicative senescence, a state of cell arrest characterized by an increased release of proinflammatory cytokines and growth factors. However, little is known about changes in the secretome profile at different stages of in vitro expansion. Some culture conditions and bioprocessing techniques have shown promise in more effectively retaining the pro-regenerative and anti-inflammatory MSC phenotype throughout expansion. Understanding how in vitro expansion conditions influence the nature and function of MSCs, and their associated secretome, may provide key insights into the underlying mechanisms driving these alterations. Elucidating the dynamic and diverse changes in the MSC secretome at each stage of in vitro expansion is a critical next step in the development of standardized, safe, and effective MSC-based therapies.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Células Madre Mesenquimatosas/metabolismo , Citocinas/metabolismo , MicroARNs/metabolismo , Diferenciación Celular , Vesículas Extracelulares/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) provide a life-long supply of haemopoietic cells and are indispensable for clinical transplantation in the treatment of malignant hematological diseases. Clinical applications require vast quantities of HSCs with maintained stemness characteristics. Meeting this demand poses often insurmountable challenges for traditional culture methods. Creating a supportive artificial microenvironment for the culture of HSCs, which allows the expansion of the cells while maintaining their stemness, is becoming a new solution for the provision of these rare multipotent HSCs. Hydrogels with good biocompatibility, excellent hydrophilicity, tunable biochemical and biophysical properties have been applied in mimicking the hematopoietic niche for the efficient expansion of HSCs. This review focuses on recent progress in the use of hydrogels in this specialized application. Advanced biomimetic strategies use for the creation of an artificial haemopoietic niche are discussed, advances in combined use of hydrogel matrices and microfluidics, including the emerging organ-on-a-chip technology, are summarized. We also provide a brief description of novel stimulus-responsive hydrogels that are used to establish an intelligent dynamic cell microenvironment. Finally, current challenges and future perspectives of engineering hydrogels for HSC biomedicine are explored.
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Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility, and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation, cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally, the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product.
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Receptores Quiméricos de Antígenos , Antígenos CD28 , Citocinas , Leucocitos Mononucleares/metabolismo , Receptores de Antígenos de Linfocitos TRESUMEN
Natural killer cells constitute a part of the innate immune system that mediates an effective immune response towards virus-infected and malignant cells. In recent years, research has focused on exploring and advancing NK cells as an active immunotherapy platform. Despite major advances, there are several key challenges that need to be addressed for the effective translation of NK cell research to clinical applications. This review highlights some of these challenges and the innovative strategies being developed to overcome them, including in vitro expansion, in vivo persistence, infiltration to the tumor site, and prevention of exhaustion.
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Inmunoterapia , Neoplasias , Humanos , Factores Inmunológicos , Células Asesinas Naturales , Neoplasias/terapiaRESUMEN
[This corrects the article DOI: 10.3389/fcell.2021.675998.].
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BACKGROUND: Regulatory T cells (Tregs) are an important cell subgroup of CD4+ T cells. Treg cells are critically involved in inducing immune tolerance, maintaining immune environment homeostasis, and preventing the occurrence of autoimmune diseases. Under normal conditions, the number of Tregs in the body is very small. This research was designed to establish an effective method to expand human peripheral blood Tregs in vitro and to analyze phenotype, purity, and function of Treg cells post-expansion. METHODS: Peripheral blood was obtained from healthy donors. CD4+CD25+CD127dim/- Treg cells were isolated from peripheral blood mononuclear cells (PBMCs) by magnetic-activated cell sorting (MACS), and an optimized culture system was used for amplification. The in vitro amplification ability of Treg cells was evaluated to determine the expression and purity of Treg cell-specific surface markers in different culture cycles. The suppressive function of Treg was determined by in vitro lymphocyte proliferation assay. RESULTS: Treg cells could be successfully isolated by magnetic activated cell sorting (MACS). After 21 days of in vitro culture, the mean expansion fold was 2,009±452.2 in ≤60 years, and there was a significant difference between the younger group and the older than 60 years group (1,238±330.0). Flow cytometry analysis revealed that the percentages of CD4+CD25+ cells and FOXP3+ cells were (93.25±3.05)% and (94.19±4.21)% on day 14, and (92.86±4.36)% and (91.55±5.62)% on day 21, respectively. In addition, the proportions of CD8+ T, CD19+ B, CD3-CD56+ natural killer cell (NK), and CD3+ CD56+ natural killer T cell (NKT) were extremely low. Lymphocyte proliferation assay demonstrated that Tregs could inhibit the proliferation of CD8+ T cells more effectively than that of CD4+ T cells. Furthermore, the suppressive capacity of Tregs was correlated with Treg-to-PBMCs ratios. CONCLUSIONS: We successfully established a technical protocol for manufacturing a large quantity of Tregs with high efficiency in vitro. The expanded Tregs have a steady FOXP3 expression and exhibited a potent immune suppression, which might have great significance in adoptive Treg therapy for treating graft-versus-host disease and autoimmune diseases.
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Ex vivo expansion of cells is necessary in regenerative medicine to generate large populations for therapeutic use. Adaptation to culture conditions prompt an increase in transcriptome diversity and decreased population heterogeneity in cKit+ cardiac interstitial cells (cCICs). The "transcriptional memory" influenced by cellular origin remained unexplored and is likely to differ between neonatal versus senescent input cells undergoing culture expansion. Transcriptional profiles derived from single cell RNASEQ platforms characterized human cCIC derived from neonatal and adult source tissue. Bioinformatic analysis revealed contrasting imprint of age influencing targets of 1) cell cycle, 2) senescence associated secretory phenotype (SASP), 3) RNA transport, and 4) ECM-receptor/fibrosis. A small subset of cCICs exist in a transcriptional continuum between "youthful" phenotype and the damaged microenvironment of LVAD tissue in which they were embedded. The connate transcriptional phenotypes offer fundamental biological insight and highlights cellular input as a consideration in culture expansion and adoptive transfer protocols.
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Senescencia Celular , Transcriptoma , Células Cultivadas , Senescencia Celular/genética , Biología Computacional , Humanos , FenotipoRESUMEN
BACKGROUND: Type 2 innate lymphoid cells (ILC2s) have emerged as key players in the development of type 2 driven diseases such as allergy and asthma. Due to their low number in the circulation, in vitro expansion is needed to unravel their mechanisms of action. PURPOSE: The aim of this study is to assess the impact of different culture conditions and address whether the method of expansion may distinctly affect healthy donor or patient-derived ILC2s. METHODS: Here, we described the impact of six different culture conditions on the proliferation, phenotype and function of human ILC2s freshly obtained from healthy donors (healthy ILC2s) and allergic patients (patient ILC2s). RESULTS: We showed that the cytokine cocktail or the PHA induced the highest proliferation of healthy ILC2s and patient ILC2s, respectively. We observed that the stromal cells OP9, used as ILC2 feeders, did not boost their proliferation, but impaired the activation marker expression and the function of patient ILC2s. Furthermore, we demonstrated that the culture conditions differently impacted the activation state of c-Kithigh and c-Kitlow ILC2s, in both healthy donors and allergic patients. Last, we also observed that ILC2s expanded only with IL-2 and IL-7 were the most prone to secrete IL-5 and IL-13 upon IL-33 stimulation. In contrast, in patients, the addition of OP9 cells during the expansion restrained their type 2 cytokine secretory functions. CONCLUSION: This report highlights that culture conditions distinctly impacted on the healthy or patient ILC2 behavior, with important consequences for their study in disease settings.
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The purpose of this work is to describe the use of Fibrin-Plasma Rich in Growth Factors (PRGF) membranes for the treatment of a rabbit alkali-burn lesion. For this purpose, an alkali-burn lesion was induced in 15 rabbits. A week later, clinical events were evaluated and rabbits were divided into five treatment groups: rabbits treated with medical treatment, with a fibrin-PRGF membrane cultured with autologous or heterologous rabbit Limbal Epithelial Progenitor Cells (LEPCs), with a fibrin-PRGF membrane in a Simple Limbal Epithelial Transplantation and with a fibrin-PRGF membrane without cultured LEPCs. After 40 days of follow-up, corneas were subjected to histochemical examination and immunostaining against corneal or conjunctival markers. Seven days after alkali-burn lesion, it was observed that rabbits showed opaque cornea, new blood vessels across the limbus penetrating the cornea and epithelial defects. At the end of the follow-up period, an improvement of the clinical parameters analyzed was observed in transplanted rabbits. However, only rabbits transplanted with cultured LEPCs were positive for corneal markers. Otherwise, rabbits in the other three groups showed positive staining against conjunctival markers. In conclusion, fibrin-PRGF membrane improved the chemically induced lesions. Nonetheless, only fibrin-PRGF membranes cultured with rabbit LEPCs were able to restore the corneal surface.
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Quemaduras Químicas , Células Epiteliales , Quemaduras Oculares , Fibrina/farmacología , Plasma , Trasplante de Células Madre , Células Madre , Animales , Autoinjertos , Quemaduras Químicas/metabolismo , Quemaduras Químicas/patología , Quemaduras Químicas/terapia , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/trasplante , Quemaduras Oculares/metabolismo , Quemaduras Oculares/patología , Quemaduras Oculares/terapia , Limbo de la Córnea/metabolismo , Limbo de la Córnea/patología , Conejos , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs with preserved myogenic properties after serial passages in vitro. Here, we describe the ice-cold treatment (ICT) method, which is a simple, economical, and efficient method for the isolation and in vitro expansion of highly pure mouse and human SCs. It involves a brief (15-30 min) incubation on ice (0 °C) of a dish containing a heterogeneous mix of adherent muscle mononuclear cells, which leads to the detachment of only the SCs, and gives rise to cultures of superior purity compared to other commonly used isolation methods. The ICT method doubles up as a gentle passaging technique, allowing SC expansion over extended periods of time without compromising their proliferation and differentiation potential. Moreover, SCs isolated and expanded using the ICT method are capable of regenerating injured muscle in vivo. The ICT method involves minimal cell manipulation, does not require any expertise or expensive reagents, it is fast, and highly reproducible, and greatly reduces the number of animals or human biopsies required in order to obtain sufficient number of SCs. The cost-effectiveness, accessibility, and technical simplicity of this method, as well as its remarkable efficiency, will no doubt accelerate SC basic and translational research bringing their therapeutic use closer to the clinic.
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Células Satélite del Músculo Esquelético , Animales , Diferenciación Celular , Proliferación Celular , Hielo , Ratones , Desarrollo de Músculos , Músculo EsqueléticoRESUMEN
Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient. Graphic abstract: Figure 1.Satellite cell isolation using the ice-cold treatment method.
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Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.
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Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclosporina/farmacología , Factor 10 de Crecimiento de Fibroblastos/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Germinativas/efectos de los fármacos , Animales , Apoptosis , Ciclo Celular , Proliferación Celular/fisiología , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Células Germinativas/fisiología , Ratones , Rolitetraciclina/farmacología , Transducción de Señal/efectos de los fármacos , Secuenciación Completa del GenomaRESUMEN
Human germ cells perpetuate human genetic and epigenetic information. However, the underlying mechanism remains elusive, due to a lack of appropriate experimental systems. Here, we show that human primordial germ cell-like cells (hPGCLCs) derived from human-induced pluripotent stem cells (hiPSCs) can be propagated to at least ~106 -fold over a period of 4 months under a defined condition in vitro. During expansion, hPGCLCs maintain an early hPGC-like transcriptome and preserve their genome-wide DNA methylation profiles, most likely due to retention of maintenance DNA methyltransferase activity. These characteristics contrast starkly with those of mouse PGCLCs, which, under an analogous condition, show a limited propagation (up to ~50-fold) and persist only around 1 week, yet undergo cell-autonomous genome-wide DNA demethylation. Importantly, upon aggregation culture with mouse embryonic ovarian somatic cells in xenogeneic-reconstituted ovaries, expanded hPGCLCs initiate genome-wide DNA demethylation and differentiate into oogonia/gonocyte-like cells, demonstrating their germline potential. By creating a paradigm for hPGCLC expansion, our study uncovers critical divergences in expansion potential and the mechanism for epigenetic reprogramming between the human and mouse germ cell lineage.
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Células Germinativas/metabolismo , Ovario/embriología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Línea Celular , Desmetilación del ADN , Metilación de ADN , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Epigenómica , Femenino , Genoma , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , RatonesRESUMEN
Cultured limbal epithelial stem cell transplantation is a clinical procedure used to regenerate the corneal epithelium in patients with limbal stem cell deficiency. The protocols used to expand limbal epithelial cells in vitro need to be optimized, since the scarcity of human ocular tissue donors is limiting the potential use of this procedure. Here, we describe a method to consecutively expand a single human limbal explant. With this method it is possible to obtain up to three limbal epithelial primary cultures from the same explant, thus increasing the efficiency of the in vitro cell culture.