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Li Y, Jin M, Gao Y, Lu L, Cao J, Liu Y, Chen Y, Wang X. Efficient establishment of an optimized culture condition for cashmere goat primary hair follicle stem cells. J Anim Sci 2023; 101:skad235. [PMID: 37429584 PMCID: PMC10370882 DOI: 10.1093/jas/skad235] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Accepted: 07/06/2023] [Indexed: 07/12/2023] Open
Abstract
Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 μM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.
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Affiliation(s)
- Yan Li
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Miaohan Jin
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Yawei Gao
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Lijin Lu
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Jing Cao
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, CAS Key Laboratory of Primate Neurobiology, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
| | - Yao Liu
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
| | - Yulin Chen
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China
| | - Xiaolong Wang
- International Joint Agriculture Research Center for Animal Bio-Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
- Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China
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Lin M, Meckes B, Chen C, Teplensky MH, Mirkin CA. Controlling Intracellular Machinery via Polymer Pen Lithography Molecular Patterning. ACS CENTRAL SCIENCE 2022; 8:1282-1289. [PMID: 36188351 PMCID: PMC9523772 DOI: 10.1021/acscentsci.2c00683] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Indexed: 06/16/2023]
Abstract
The plasma membrane and the actomyosin cytoskeleton play key roles in controlling how cells sense and interact with their surrounding environment. Myosin, a force-generating actin network-associated protein, is a major regulator of plasma membrane tension, which helps control endocytosis. Despite the important link between plasma membranes and actomyosin (the actin-myosin complex), little is known about how the actomyosin arrangement regulates endocytosis. Here, nanoscopic ligand arrangements defined by polymer pen lithography (PPL) are used to control actomyosin contractility and examine cell uptake. Confocal microscopy, atomic force microscopy, and flow cytometry suggest that the cytoskeletal tension imposed by the nanoscopic ligand arrangement can actively regulate cellular uptake through clathrin- and caveolin-mediated pathways. Specifically, ligand arrangements that increase cytoskeletal tension tend to reduce the cellular uptakes of cholera toxin (CTX) and spherical nucleic acids (SNAs) by regulating endocytic budding and limiting the formation of clathrin- and caveolae-coated pits. Collectively, this work demonstrates how the cell endocytic fate is regulated by actomyosin mechanical forces, which can be tuned by subcellular cues defined by PPL.
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Affiliation(s)
- Millicent Lin
- Department
of Biomedical Engineering, Northwestern
University, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- International
Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
| | - Brian Meckes
- International
Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- Department
of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
| | - Chaojian Chen
- International
Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- Department
of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
| | - Michelle H. Teplensky
- International
Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- Department
of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
| | - Chad A. Mirkin
- Department
of Biomedical Engineering, Northwestern
University, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- International
Institute for Nanotechnology, 2145 Sheridan Road, Evanston, Illinois 60208, United
States
- Department
of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States
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Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing. Cornea 2021; 40:203-214. [PMID: 33086282 DOI: 10.1097/ico.0000000000002560] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Accepted: 08/26/2020] [Indexed: 12/13/2022]
Abstract
PURPOSE To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. METHODS UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. RESULTS Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. CONCLUSIONS Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.
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Liu P, Chen S, Wang Y, Chen X, Guo Y, Liu C, Wang H, Zhao Y, Wu D, Shan Y, Zhang J, Wu C, Li D, Zhang Y, Zhou T, Chen Y, Liu X, Li C, Wang L, Jia B, Liu J, Feng B, Cai J, Pei D. Efficient induction of neural progenitor cells from human ESC/iPSCs on Type I Collagen. SCIENCE CHINA-LIFE SCIENCES 2021; 64:2100-2113. [PMID: 33740188 DOI: 10.1007/s11427-020-1897-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 01/19/2021] [Indexed: 10/21/2022]
Abstract
A stable, rapid and effective neural differentiation method is essential for the clinical applications of human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in treating neurological disorders and diseases. Herein, we established a novel and robust monolayer differentiation method to produce functional neural progenitor cells (NPCs) from human ESC/iPSCs on Type I Collagen. The derived cells not only displayed the requisite markers, but also behaved similarly to classic NPCs both in vitro and in vivo. Upon transplantation into traumatic brain injury model, the derived NPCs facilitated recovery from injury. We also found that SMAD signaling stayed down throughout the differentiation process on Type I Collagen, and the pluripotent signals were rapidly downregulated along with raising up of neural early markers on the third day. Meanwhile, ATAC-seq data showed the related mediation of distinct transcriptome and global chromatin dynamics during NPC induction. Totally, our results thus provide a convenient way to generate NPCs from human ESC/iPSCs for neural diseases' treatment.
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Affiliation(s)
- Pengfei Liu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Ambulatory Surgical Center, The 2nd Clinical medical College (Shenzhen People's Hospital) of Jinan University, The 1st Affiliated Hospitals of Southern University of Science and Technology, Shenzhen, 518020, China.,Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou, 510632, China
| | - Shubin Chen
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Yaofeng Wang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China.,School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China.,Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, China
| | - Xiaoming Chen
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Yiping Guo
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Chunhua Liu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Haitao Wang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yifan Zhao
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China.,Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Changchun, 130012, China
| | - Di Wu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Changchun, 130012, China
| | - Yongli Shan
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Jian Zhang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Chuman Wu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Dongwei Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yanmei Zhang
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China
| | - Tiancheng Zhou
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China
| | - Yaoyu Chen
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Changchun, 130012, China
| | - Xiaobo Liu
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Changchun, 130012, China
| | - Chenxu Li
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.,Department of Regenerative Medicine, School of Pharmaceutical Science, Jilin University, Changchun, 130012, China
| | - Lihui Wang
- Department of Pathology, Medical College, Jinan University, Guangzhou, 510632, China
| | - Bei Jia
- The Center for Prenatal and Hereditary Disease Diagnosis, Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Jie Liu
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China
| | - Bo Feng
- School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China
| | - Jinglei Cai
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China. .,Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, China. .,Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
| | - Duanqing Pei
- CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China. .,Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510005, China. .,Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Hong Kong SAR, China. .,Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, 100101, China.
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Yang S, Xin C, Zhang B, Zhang H, Hao Y. Synergistic effects of Rho kinase inhibitor Y-27632 and Noggin overexpression on the proliferation and neuron-like cell differentiation of stem cells derived from human exfoliated deciduous teeth. IUBMB Life 2019; 72:665-676. [PMID: 31889420 DOI: 10.1002/iub.2208] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Accepted: 11/22/2019] [Indexed: 12/22/2022]
Abstract
Stem cells from human exfoliated deciduous teeth (SHEDs) are highly proliferative, clonogenic, and multipotent stem cells with a neural crest cell origin. This property could be a desirable option for potential therapeutic applications. In this study, we focus on the effects of Rho kinase inhibitors Y-27632 and Noggin on the proliferation of SHEDs and their differentiation into neuron-like cells. SHEDs were extracted from 10 samples of deciduous teeth obtained from healthy children aged from 5 to 10. The passaged SHEDs were transfected with Noggin, Y-27632, or their combination. By means of MTT and colony formation assays, the effects of Y-27632 and Noggin on cell viability and colony formation were detected. Cellular morphology and neurosphere formation were observed under a microscope. Y-27632 transfection in SHEDs showed enhanced cell viability, colony formation, and neurosphere formation indicating that Y-27632 could promote cell proliferation of SHEDs. Furthermore, we observed that the SHEDs treated with Noggin in combination with Y-27632 displayed typical neuron-like cell morphology and reticular processes. Noggin or Y-27632 alone or in combination induced obviously increased NSE, Nestin, and GFAP levels, which were highest in SHEDs treated with the combination of Noggin and Y-27632. These findings suggest that Y-27632 promotes the proliferation of SHEDs, and Y-27632 and Noggin in combination have a synergistic effect on promoting differentiation of SHEDs into neuron-like cells.
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Affiliation(s)
- Si Yang
- Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, China
| | - Cuijuan Xin
- Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, China
| | - Bo Zhang
- Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, China
| | - Hongbo Zhang
- Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, China
| | - Yunpeng Hao
- Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, China
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Protein Kinases and Their Inhibitors in Pluripotent Stem Cell Fate Regulation. Stem Cells Int 2019; 2019:1569740. [PMID: 31428157 PMCID: PMC6681599 DOI: 10.1155/2019/1569740] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Revised: 05/31/2019] [Accepted: 06/16/2019] [Indexed: 12/25/2022] Open
Abstract
Protein kinases modulate the reversible postmodifications of substrate proteins to their phosphorylated forms as an essential process in regulating intracellular signaling transduction cascades. Moreover, phosphorylation has recently been shown to tightly control the regulatory network of kinases responsible for the induction and maintenance of pluripotency, defined as the particular ability to differentiate pluripotent stem cells (PSCs) into every cell type in the adult body. In particular, emerging evidence indicates that the balance between the self-renewal and differentiation of PSCs is regulated by the small molecules that modulate kinase signaling pathways. Furthermore, new reprogramming technologies have been developed using kinase modulators, which have provided novel insight of the mechanisms underlying the kinase regulatory networks involved in the generation of induced pluripotent stem cells (iPSCs). In this review, we highlight the recent progress made in defining the roles of protein kinase signaling pathways and their small molecule modulators in regulating the pluripotent states, self-renewal, reprogramming process, and lineage differentiation of PSCs.
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Weng NJH, Cheung C, Talbot P. Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling. Stem Cell Res 2018; 33:233-246. [PMID: 30458343 PMCID: PMC6414319 DOI: 10.1016/j.scr.2018.10.022] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/17/2016] [Revised: 10/25/2018] [Accepted: 10/31/2018] [Indexed: 12/11/2022] Open
Abstract
This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.
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Affiliation(s)
- Nikki Jo-Hao Weng
- Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA 92521, United States; Cell Molecular and Developmental Biology Graduate Program, University of California, Riverside, CA 92521, United States
| | - Cindy Cheung
- Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA 92521, United States
| | - Prue Talbot
- Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA 92521, United States; Cell Molecular and Developmental Biology Graduate Program, University of California, Riverside, CA 92521, United States.
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8
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Biochemical re-programming of human dermal stem cells to neurons by increasing mitochondrial membrane potential. Cell Death Differ 2018; 26:1048-1061. [PMID: 30154448 DOI: 10.1038/s41418-018-0182-8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 07/15/2018] [Accepted: 07/22/2018] [Indexed: 01/07/2023] Open
Abstract
Stem cells are generally believed to contain a small number of mitochondria, thus accounting for their glycolytic phenotype. We demonstrate here, however, that despite an indispensable glucose dependency, human dermal stem cells (hDSCs) contain very numerous mitochondria. Interestingly, these stem cells segregate into two distinct subpopulations. One exhibits high, the other low-mitochondrial membrane potentials (Δψm). We have made the same observations with mouse neural stem cells (mNSCs) which serve here as a complementary model to hDSCs. Strikingly, pharmacologic inhibition of phosphoinositide 3-kinase (PI3K) increased the overall Δψm, decreased the dependency on glycolysis and led to formation of TUJ1 positive, electrophysiologically functional neuron-like cells in both mNSCs and hDSCs, even in the absence of any neuronal growth factors. Furthermore, of the two, it was the Δψm-high subpopulation which produced more mitochondrial reactive oxygen species (ROS) and showed an enhanced neuronal differentiation capacity as compared to the Δψm-low subpopulation. These data suggest that the Δψm-low stem cells may function as the dormant stem cell population to sustain future neuronal differentiation by avoiding excessive ROS production. Thus, chemical modulation of PI3K activity, switching the metabotype of hDSCs to neurons, may have potential as an autologous transplantation strategy for neurodegenerative diseases.
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9
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Kamishibahara Y, Kawaguchi H, Shimizu N. Rho kinase inhibitor Y-27632 promotes neuronal differentiation in mouse embryonic stem cells via phosphatidylinositol 3-kinase. Neurosci Lett 2016; 615:44-9. [PMID: 26797580 DOI: 10.1016/j.neulet.2016.01.022] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Revised: 01/14/2016] [Accepted: 01/14/2016] [Indexed: 10/22/2022]
Abstract
Rho kinase (ROCK) regulates the functions of several target proteins via its kinase activity. Therefore, ROCK activity inhibition may provide new possibilities of controlling the in vitro neuronal differentiation of embryonic stem (ES) cells. When we investigated the effects of the ROCK inhibitor Y-27632 on ES cell differentiation, we found that this inhibitor promoted the differentiation of these cells into neurons. Furthermore, we found that ROCK inhibition may promote the neuronal differentiation of ES cells by activating extracellular signal-regulated kinase (ERK) involved in the ERK signaling pathway. In this study, we investigated the effects of specific inhibitors of several cellular signaling components on the promotion of neuronal differentiation in ES cells to clarify the roles of cellular signaling pathways in the ROCK inhibitor-mediated cell differentiation process. Our results suggest that ERK may be activated via the Ras/Raf/MEK, the PI3K/PKC, or the Cdc42/Rac signaling pathways in the ROCK inhibitor-mediated promotion of neuronal differentiation in ES cells.
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Affiliation(s)
- Yu Kamishibahara
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan.
| | - Hideo Kawaguchi
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan.
| | - Norio Shimizu
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan; Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama 350-8585, Japan.
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10
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Chen L, Wang GD, Liu JP, Wang HS, Liu XM, Wang Q, Cai XH. miR-135a modulates tendon stem/progenitor cell senescence via suppressing ROCK1. Bone 2015; 71:210-6. [PMID: 25460182 DOI: 10.1016/j.bone.2014.11.001] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2014] [Revised: 10/29/2014] [Accepted: 11/02/2014] [Indexed: 12/23/2022]
Abstract
Tendon stem/progenitor cell (TSPC) senescence may lead to age-related tendon disorders and impair tendon regeneration and replacement capacity in humans. However, the mechanisms governing TSPC aging and degeneration remain obscure. Recently, it has been reported that Rho-associated coiled-coil protein kinase 1 (ROCK1) might be a key player in TSPC aging process. miRNAs are also involved in cellular senescence. In this study, whether miRNAs modulate senescence of TSPCs through targeting ROCK1 was evaluated. We found that miR-135a, which directly binds to the 3'-untranslated region of ROCK1, is significantly downregulated in aged compared with young TSPCs. Overexpression of miR-135a in young TSPCs suppresses senescence, promotes proliferation, and induces migration and tenogenic differentiation, whereas suppression of miR-135a in aged TSPCs has the opposite effects. By gain-of-function and loss-of-function studies, we confirmed that ROCK1 mediates the effects of miR-135a in TSPCs. Taken together, our data suggest that miR-135a plays an important role in TSPC senescence via targeting ROCK1.
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Affiliation(s)
- Lei Chen
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China
| | - Guo-Dong Wang
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China
| | - Jun-Peng Liu
- Department of Orthopaedics Surgery, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China
| | - Hua-Song Wang
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China
| | - Xi-Ming Liu
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China
| | - Qing Wang
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China.
| | - Xian-Hua Cai
- Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan 430000, PR China.
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Otsu M, Nakayama T, Inoue N. Pluripotent stem cell-derived neural stem cells: From basic research to applications. World J Stem Cells 2014; 6:651-657. [PMID: 25426263 PMCID: PMC4178266 DOI: 10.4252/wjsc.v6.i5.651] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2014] [Revised: 09/04/2014] [Accepted: 09/17/2014] [Indexed: 02/07/2023] Open
Abstract
Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.
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Nakamura M, Kamishibahara Y, Kitazawa A, Kawaguchi H, Shimizu N. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons. Cytotechnology 2014; 68:409-17. [PMID: 25354731 DOI: 10.1007/s10616-014-9792-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2014] [Accepted: 09/27/2014] [Indexed: 10/24/2022] Open
Abstract
Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells.
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Affiliation(s)
- Mai Nakamura
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan
| | - Yu Kamishibahara
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan
| | - Ayako Kitazawa
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan.,Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama, 350-8585, Japan
| | - Hideo Kawaguchi
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan
| | - Norio Shimizu
- Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, Gunma, 374-0193, Japan. .,Bio-Nano Electronics Research Center, Toyo University, 2100 Kujirai, Kawagoe-shi, Saitama, 350-8585, Japan.
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