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1.
Cells Tissues Organs ; 2022 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-35901725

RESUMEN

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel and lapatinib was nontoxic at 0.01, 0.01, and 0.2µM concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

2.
Stem Cell Rev Rep ; 18(7): 2279-2295, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35175538

RESUMEN

BACKGROUND: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). METHODS: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). RESULTS: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. CONCLUSION: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Mesenquimatosas , Médula Ósea , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos
3.
Cell Tissue Res ; 386(2): 321-333, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34319434

RESUMEN

Human otic organoids generated from pluripotent stem cells (PSCs) provide a promising platform for modeling, drug testing, and cell-based therapies of inner ear diseases. However, providing the appropriate niche that resembles inner ear development and its vasculature to generate otic organoids is less conspicuous. Here, we devised a strategy to enhance maturation of otic progenitor cells toward human hair cell-like cells (HCLCs) by assembling three-dimensional (3D) otic organoids that contain human PSC-derived otic cells, endothelial cells, and mesenchymal stem cells (MSCs). Heterotopic implantation of otic organoids, designated as grafted otic organoids (GOs), in ex ovo chick embryo chorioallantoic membrane (CAM) stimulated maturation of the HCLCs. Functional analysis revealed the presence of voltage-gated potassium currents without detectable sodium currents in these cells in the GOs. Our results demonstrated that implantation of 3D heterotypic cell mixtures of otic organoids improved maturation of human HCLCs. This GO-derived HCLCs could be an attractive source for drug discovery and other biomedical applications.


Asunto(s)
Células Ciliadas Auditivas/citología , Organoides/citología , Células Madre Pluripotentes/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Embrión de Pollo , Oído Interno/citología , Humanos
4.
Stem Cell Rev Rep ; 17(6): 1975-1992, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34115316

RESUMEN

INTRODUCTION: Pluripotent stem cells (PSCs) are promising tools for modern regenerative medicine applications because of their stemness properties, which include unlimited self-renewal and the ability to differentiate into all cell types in the body. Evidence suggests that a rare population of cells within a tumor, termed cancer stem cells (CSCs), exhibit stemness and phenotypic plasticity properties that are primarily responsible for resistance to chemotherapy, radiotherapy, metastasis, cancer development, and tumor relapse. Different therapeutic approaches that target CSCs have been developed for tumor eradication. RESULTS AND DISCUSSION: In this review, we first provide an overview of different viewpoints about the origin of CSCs. Particular attention has been paid to views believe that CSCs are probably appeared through dysregulation of very small embryonic-like stem cells (VSELs) which reside in various tissues as the main candidate for tissue-specific stem cells. The expression of pluripotency markers in these two types of cells can strengthen the validity of this theory. In this regard, we discuss the common properties of CSCs and PSCs, and highlight the potential of PSCs in cancer studies, therapeutic applications, as well as educating the immune system against CSCs. CONCLUSION: In conclusion, the resemblance of CSCs to PSCs can provide an appropriate source of CSC-specific antigens through cultivation of PSCs which brings to light promising ideas for prophylactic and therapeutic cancer vaccine development.


Asunto(s)
Neoplasias , Células Madre Pluripotentes , Células Madre Embrionarias/metabolismo , Humanos , Neoplasias/patología , Células Madre Neoplásicas/metabolismo , Células Madre Pluripotentes/metabolismo , Vacunación
5.
Stem Cells Dev ; 30(8): 428-440, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33787359

RESUMEN

Directed differentiation of human pluripotent stem cells (hPSCs) uses a growing number of small molecules and growth factors required for in vitro generation of renal lineage cells. Although current protocols are relatively inefficient or expensive. The first objective of the present work was to establish a new differentiation protocol for generating renal precursors. We sought to determine if inducer of definitive endoderm 1 (IDE1), a cost-effective small molecule, can be used to replace activin A. Gene expression data showed significantly increased expressions of nephrogenic markers in cells differentiated with 20 nM IDE1 compared with cells differentiated with activin A. Thus, renal lineage cells could be generated by this alternative approach. Afterward, we determined whether coculture of endothelial and mesenchymal cells could increase the maturation of three-dimensional (3D) renal structures. For this purpose, we employed a 3D coculture system in which hPSC-derived kidney precursors were cocultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs), hereafter named RMEM (renal microtissue derived from coculture of renal precursors with endothelial and mesenchymal stem cells). hPSC-derived kidney precursors were cultured either alone [renal microtissue (RM)] or in coculture with human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells at an approximate ratio of 10:7:2, respectively. Immunofluorescent staining showed expressions of kidney-specific markers synaptopodin, LTL, and E-cadherin, as well as CD31+ ECs that were distributed throughout the RMEMs. Quantitative real-time polymerase chain reaction analysis confirmed a significant increase in gene expressions of the renal-specific markers in RMEMs compared with RMs. These findings demonstrated that renal precursors cocultured with endothelial and MSCs showed greater maturity compared with RMs. Moreover, ex ovo transplantation induced further maturation in the RMEM constructs. Our novel approach enabled the generation of RMEM that could potentially be used in high-throughput drug screening and nephrotoxicology studies.


Asunto(s)
Diferenciación Celular/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Riñón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Pluripotentes/metabolismo , Línea Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Inmunohistoquímica , Riñón/citología , Células Madre Mesenquimatosas/citología , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
Stem Cell Reports ; 16(1): 39-55, 2021 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-33357408

RESUMEN

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.


Asunto(s)
Embrión de Pollo/metabolismo , Quimerismo/veterinaria , Células Madre Pluripotentes/trasplante , Animales , Diferenciación Celular , Linaje de la Célula , Embrión de Pollo/citología , Pollos , Desarrollo Embrionario , Edición Génica , Humanos , Proteínas con Homeodominio LIM/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
7.
Cells Tissues Organs ; 206(6): 317-329, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-31340210

RESUMEN

Burn wound treatment is difficult and one of the most challenging problems in the clinic. Researchers have examined the applications of mesenchymal stem cells as a cell-based therapy for skin regeneration. But the role of human bone marrow mesenchymal stem cell conditioned medium (hBM-MSC-CM) in the treatment of burn injury remains unclear. This research aims at detecting whether hBM-MSC-CM can increase the wound healing of deep second-degree burns in male rats. In this study, 32 adult male rats per each time point were randomly divided into four groups: (1) control group, (2) sham group (DMEM), (3) common treatment group (CT), and (4) conditioned media group (CM). A 3 × 3 cm circular burn was created on the back of the rats. On postsurgical days 7, 15, and 28, the wound closure area of each wound was measured and then the skin samples were removed and analyzed using stereological methods. Wound closure area was significantly increased in the CM and CT groups on the 15th and the 28th day after burn injury compared to the control and DMEM groups. The stereological parameters and immunohistochemistry analysis of the wounds revealed significantly improved healing in the CM group compared to the control and other groups. It is concluded that these findings indicate that hBM-MSC-CM promotes skin wound healing by increasing cell proliferation, regulating collagen synthesis and collagen composition, and inducing angiogenesis at the injury site.

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