scholarly journals Hypoxia-preconditioned adipose-derived stem cells combined with scaffold promote urethral reconstruction by upregulation of angiogenesis and glycolysis

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiang Wan ◽  
Min-kai Xie ◽  
Huan Xu ◽  
Zi-wei Wei ◽  
Hai-jun Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Umbilical Vein ◽  
Urethral Reconstruction ◽  
Therapeutic Effects ◽  
Adipose Derived Stem Cells ◽  
Promising Alternative ◽  
Hypoxia Preconditioning ◽  
Novel Scaffold

Abstract Rationale Tissue engineering is a promising alternative for urethral reconstruction, and adipose-derived stem cells (ADSCs) are widely used as seeding cells. Hypoxia preconditioning can significantly enhance the therapeutic effects of ADSCs. The low oxygen tension of postoperative wound healing is inevitable and may facilitate the nutritional function of ADSCs. This study aimed to investigate if hypoxia-preconditioned ADSCs, compared to normoxia-preconditioned ADSCs, combined with scaffold could better promote urethral reconstruction and exploring the underlying mechanism. Methods In vitro, paracrine cytokines and secretomes that were secreted by hypoxia- or normoxia-preconditioned ADSCs were added to cultures of human umbilical vein endothelial cells (HUVECs) to measure their functions. In vivo, hypoxia- or normoxia-preconditioned ADSCs were seeded on a porous nanofibrous scaffold for urethral repair on a defect model in rabbits. Results The in vitro results showed that hypoxia could enhance the secretion of VEGFA by ADSCs, and hypoxia-preconditioned ADSCs could enhance the viability, proliferation, migration, angiogenesis, and glycolysis of HUVECs (p < 0.05). After silencing VEGFA, angiogenesis and glycolysis were significantly inhibited (p < 0.05). The in vivo results showed that compared to normoxia-preconditioned ADSCs, hypoxia-preconditioned ADSCs combined with scaffolds led to a larger urethral lumen diameter, preserved urethral morphology, and enhanced angiogenesis (p < 0.05). Conclusions Hypoxia preconditioning of ADSCs combined with scaffold could better promote urethral reconstruction by upregulating angiogenesis and glycolysis. Hypoxia-preconditioned ADSCs combined with novel scaffold may provide a promising alternative treatment for urethral reconstruction.

scholarly journals Hypoxia-preconditioned adipose-derived stem cells combined with scaffold promote urethral reconstruction by up-regulation of angiogenesis and glycolysis

2020 ◽  
Author(s):  
Xiang Wan ◽  
Min-kai Xie ◽  
Huan Xu ◽  
Zi-wei Wei ◽  
Hai-jun Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Umbilical Vein ◽  
Urethral Reconstruction ◽  
Therapeutic Effects ◽  
Adipose Derived Stem Cells ◽  
Promising Alternative ◽  
Hypoxia Preconditioning ◽  
Novel Scaffold

Abstract Rationale: Tissue engineering is a promising alternative for urethral reconstruction, and adipose-derived stem cells (ADSCs) are widely used as seeding cells. Hypoxia preconditioning can significantly enhance the therapeutic effects of ADSCs. The low oxygen tension of postoperative wound healing is inevitable and may facilitate the nutritional function of ADSCs. This study aimed to investigate if hypoxia preconditioned ADSCs, compared to normxia preconditioned ADSCs, combined with scaffold could better promote urethral reconstruction and exploring the underlying mechanism.Methods: In vitro, paracrine cytokines and secretomes that were secreted by hypoxia- or normoxia-preconditioned ADSCs were added to cultures of human umbilical vein endothelial cells (HUVECs) to measure their functions. In vivo, hypoxia- or normoxia-preconditioned ADSCs were seeded on a porous nanofibrous scaffold for urethral repair on a defect model in rabbits.Results: The in vitro results showed that hypoxia could enhance the secretion of VEGFA by ADSCs, and hypoxia-preconditioned ADSCs could enhance the viability, proliferation, migration, angiogenesis and glycolysis of HUVECs (p < 0.05). After silencing VEGFA, angiogenesis and glycolysis were significantly inhibited (p < 0.05). The in vivo results showed that compared to normoxia-preconditioned ADSCs, hypoxia-preconditioned ADSCs combined with scaffolds led to a larger urethral lumen diameter, preserved urethral morphology and enhanced angiogenesis (p < 0.05). Conclusions: Hypoxia preconditioning of ADSCs combined with scaffold could better promote urethral reconstruction by upregulating angiogenesis and glycolysis. Hypoxia-preconditioned ADSCs combined with novel scaffold may provide a promising alternative treatment for urethral reconstruction.

scholarly journals Hypoxia-Preconditioned Adipose-Derived Stem Cells Combined with Scaffold Promote Urethral Reconstruction by Up-Regulation of Angiogenesis and Glycolysis

2020 ◽  
Author(s):  
Xiang Wan ◽  
Min-kai Xie ◽  
Huan Xu ◽  
Zi-wei Wei ◽  
Hai-jun Yao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Umbilical Vein ◽  
Urethral Reconstruction ◽  
Therapeutic Effects ◽  
Adipose Derived Stem Cells ◽  
Promising Alternative ◽  
Hypoxia Preconditioning ◽  
Novel Scaffold

Abstract Rationale: Tissue engineering is a promising alternative for urethral reconstruction, and adipose-derived stem cells (ADSCs) are widely used as seeding cells. Hypoxia preconditioning can significantly enhance the therapeutic effects of ADSCs. The low oxygen tension of postoperative wound healing is inevitable and may facilitate the nutritional function of ADSCs. This study aimed to investigate if hypoxia preconditioned ADSCs, compared to normxia preconditioned ADSCs, combined with scaffold could better promote urethral reconstruction and exploring the underlying mechanism.Methods: In vitro, paracrine cytokines and secretomes that were secreted by hypoxia- or normoxia-preconditioned ADSCs were added to cultures of human umbilical vein endothelial cells (HUVECs) to measure their functions. In vivo, hypoxia- or normoxia-preconditioned ADSCs were seeded on a porous nanofibrous scaffold for urethral repair on a defect model in rabbits.Results: The in vitro results showed that hypoxia could enhance the secretion of VEGFA by ADSCs, and hypoxia-preconditioned ADSCs could enhance the viability, proliferation, migration, angiogenesis and glycolysis of HUVECs (p < 0.05). After silencing VEGFA, angiogenesis and glycolysis were significantly inhibited (p < 0.05). The in vivo results showed that compared to normoxia-preconditioned ADSCs, hypoxia-preconditioned ADSCs combined with scaffolds led to a larger urethral lumen diameter, preserved urethral morphology and enhanced angiogenesis (p < 0.05). Conclusions: Hypoxia preconditioning of ADSCs combined with scaffold could better promote urethral reconstruction by upregulating angiogenesis and glycolysis. Hypoxia-preconditioned ADSCs combined with novel scaffold may provide a promising alternative treatment for urethral reconstruction.

scholarly journals Engineered adipose-derived stem cells with IGF-1-modified mRNA ameliorates osteoarthritis development

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Haoyu Wu ◽  
Zhi Peng ◽  
Ying Xu ◽  
Zixuan Sheng ◽  
Yanshan Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Efficiency ◽  
Therapeutic Potential ◽  
Immunohistochemical Analysis ◽  
Fluorescent Labeling ◽  
Knee Joints ◽  
Adipose Derived Stem Cells ◽  
Promising Alternative

Abstract Background Osteoarthritis (OA), a prevalent degenerative disease characterized by degradation of extracellular matrix (ECM), still lacks effective disease-modifying therapy. Mesenchymal stem cells (MSCs) transplantation has been regarded as the most promising approach for OA treatment while engrafting cells alone might not be adequate for effective regeneration. Genetic modification has been used to optimize MSC-based therapy; however, there are still significant limitations that prevent the clinical translation of this therapy including low efficacy and safety concerns. Recently, chemically modified mRNA (modRNA) represents a promising alternative for the gene-enhanced MSC therapy. In this regard, we hypothesized that adipose derived stem cells (ADSCs) engineered with modRNA encoding insulin-like growth factor 1 (IGF-1) were superior to native ADSCs on ameliorating OA development. Methods Mouse ADSCs were acquired from adipose tissue and transfected with modRNAs. First, the kinetics and efficacy of modRNA-mediated gene transfer in mouse ADSCs were analyzed in vitro. Next, we applied an indirect co-culture system to analyze the pro-anabolic potential of IGF-1 modRNA engineered ADSCs (named as IGF-1-ADSCs) on chondrocytes. Finally, we evaluated the cell retention and chondroprotective effect of IGF-1-ADSCs in vivo using fluorescent labeling, histology and immunohistochemistry. Results modRNA transfected mouse ADSCs with high efficiency (85 ± 5%) and the IGF-1 modRNA-transfected ADSCs facilitated burst-like production of bio-functional IGF-1 protein. In vitro, IGF-1-ADSCs induced increased anabolic markers expression of chondrocytes in inflammation environment compared to untreated ADSCs. In a murine OA model, histological and immunohistochemical analysis of knee joints harvested at 4 weeks and 8 weeks after OA induction suggested IGF-1-ADSCs had superior therapeutic effect over native ADSCs demonstrated by lower histological OARSI score and decreased loss of cartilage ECM. Conclusions These findings collectively supported the therapeutic potential of IGF-1-ADSCs for clinical OA management and cartilage repair.

scholarly journals The CNGRCLLII(KLAKLAK)2 peptide shows cytotoxicity against HUVECs by inducing apoptosis: An in vitro and in vivo study

Tumor Biology ◽  
2017 ◽  
Vol 39 (5) ◽  
pp. 101042831770164
Author(s):  
Guanying Wang ◽  
Na Yuan ◽  
Shangke Huang ◽  
Lu Feng ◽  
Rui Han ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Adipose Derived Stem Cells ◽  
Human Umbilical Vein ◽  
Tumor Tissues ◽  
Umbilical Vein Endothelial Cells

Fibrinogen Asn-Gly-Arg motif can specifically recognize and bind to Aminopeptidase N (CD13) on vascular endothelial cells in newly formed tumor vessels. Adipose-derived stem cells can serve as ideal vectors for gene therapy because of their ability of migrating to tumor tissues. First, this study was aimed to design a new peptide (CNGRCLLII(KLAKLAK)2) named CNAK which contains cyclic Asn-Gly-Arg motif and test its biological activity against human umbilical vein endothelial cells. Second, we aimed to construct stably transfected adipose-derived stem cells which express the CNAK peptide and investigate their anti-angiogenic activity in vivo. Adipose-derived stem cells were employed to localize CNAK on vascular endothelial cells in tumors based on their homing property. First of all, the new peptide was synthesized, which effectively entered into CD13+ human umbilical vein endothelial cells and showed cytotoxicity against human umbilical vein endothelial cells. The peptide induced apoptosis of human umbilical vein endothelial cells in a time- and dose-dependent manner, inhibited the expression of Bcl-2, and promoted the expression of Caspase-3 in human umbilical vein endothelial cells. Furthermore, the migration and tube formation of human umbilical vein endothelial cells were inhibited by CNAK. Primary adipose-derived stem cells were then isolated and identified. Stably transfected adipose-derived stem cells which express CNAK peptide (CNAK-ASCs) were successfully established, and the migration of CNAK-ASCs was assessed. In vivo, CNAK-ASCs were found to inhibit the growth and angiogenesis of breast cancer xenografts. This effect may be through inhibiting the secretion of matrix metalloproteinase-2 and membrane type 1-matrix metalloproteinase in vivo. It was also found that CNAK-ASCs reduced the quantity of breast cancer stem cells in tumor tissues. Our data suggested that the new peptide CNAK containing Asn-Gly-Arg motif had anti-angiogenic activity in vitro and in vivo.

scholarly journals Exosomes Secreted from Hypoxia-Preconditioned Mesenchymal Stem Cells Prevent Steroid-Induced Osteonecrosis of the Femoral Head by Promoting Angiogenesis in Rats

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Na Yuan ◽  
Zhaogang Ge ◽  
Wenchen Ji ◽  
Jia Li
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Femoral Head ◽  
Bone Loss ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Therapeutic Effects ◽  
Umbilical Vein Endothelial Cells

Recent studies have suggested that exosomes exert similar therapeutic effects to those of mesenchymal stem cells (MSCs) in regenerative medicine and MSCs-derived exosomes exhibit therapeutic effects on steroid-induced osteonecrosis of the femoral head (ONFH). Furthermore, reparative functions of exosomes from MSCs are enhanced by hypoxia treatment of the cells. However, there are no related reports about whether exosomes derived from hypoxia-preconditioned MSCs could show better therapeutic effects on steroid-induced ONFH. In vitro, we investigated the effects of hypoxia precondition on exosomes derived from bone marrow mesenchymal stem cells (BMMSCs) from rats and the proangiogenic ability of exosomes derived from hypoxia-preconditioned BMMSCs. In vivo, we investigated the role of exosomes from hypoxia-preconditioned BMMSCs on angiogenesis and protecting osteonecrosis in a rat ONFH model. We found that the potential of the proangiogenic ability of exosomes derived from hypoxia-preconditioned BMMSCs was higher than exosomes derived from BMMSCs cultured under normoxia. Exosomes derived from hypoxia-preconditioned BMMSCs significantly promoted proliferation, migration, vascular endothelial growth factor (VEGF) expression, and tube formation of human umbilical vein endothelial cells (HUVECs) compared with exosomes derived from BMMSCs cultured under normoxia. Administration of exosomes derived from hypoxia-preconditioned BMMSCs significantly prevented bone loss and increased vessel volume in the femoral head compared with exosomes derived from BMMSCs cultured under normoxia. Taken together, our data suggest that exosomes derived from hypoxia-preconditioned BMMSCs exert better therapeutic effects on steroid-induced ONFH by promoting angiogenesis and preventing bone loss.

scholarly journals WTD Attenuating Rheumatoid Arthritis via Suppressing Angiogenesis and Modulating the PI3K/AKT/mTOR/HIF-1α Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Xin Ba ◽  
Ying Huang ◽  
Pan Shen ◽  
Yao Huang ◽  
Hui Wang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Cell Proliferation ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Migration And Invasion ◽  
Network Pharmacology ◽  
Therapeutic Effects ◽  
Promising Alternative

Background: Wutou Decoction (WTD), as a classic prescription, has been generally used to treat rheumatoid arthritis (RA) for two thousand years in China. However, the potential protective effects of WTD on rheumatoid arthritis and its possible mechanism have rarely been reported.Purpose: The aim of this study was to explore the possible mechanism of WTD against RA and a promising alternative candidate for RA therapy.Methods: A model of collagen-induced arthritis (CIA) was constructed in rats to assess the therapeutic effects of WTD. Histopathological staining, immunofluorescence, and western blotting of synovial sections were conducted to detect the antiangiogenic effects of WTD. Then, cell viability assays, flow cytometry, scratch healing assays, and invasion assays were conducted to explore the effects of WTD on MH7A human fibroblast-like synoviocyte (FLS) cell proliferation, apoptosis, migration, and invasion in vitro. The ability of WTD to induce blood vessel formation after MH7A cell and human umbilical vein endothelial cell line (HUVEC) coculture with WTD intervention was detected by a tube formation assay. The mechanisms of WTD were screened by network pharmacology and confirmed by in vivo and in vitro experiments.Results: WTD ameliorated the symptoms and synovial pannus hyperplasia of CIA rats. Treatment with WTD inhibited MH7A cell proliferation, migration, and invasion and promoted MH7A apoptosis. WTD could inhibit MH7A cell expression of proangiogenic factors, including VEGF and ANGI, to induce HUVEC tube formation. Furthermore, the PI3K-AKT-mTOR-HIF-1α pathway was enriched as a potential target of WTD for the treatment of RA through network pharmacology enrichment analysis. Finally, it was confirmed in vitro and in vivo that WTD inhibits angiogenesis in RA by interrupting the PI3K-AKT-mTOR-HIF-1α pathway.Conclusion: WTD can inhibit synovial hyperplasia and angiogenesis, presumably by inhibiting the migration and invasion of MH7A cells and blocking the production of proangiogenic effectors in MH7A cells. The possible underlying mechanism by which WTD ameliorates angiogenesis in RA is the PI3K-AKT-mTOR-HIF-1α pathway.

Abstract 209: Human Umbilical Vein Endothelial Cell Conditioned Medium in the Presence of High Glucose Increases in vitro Adipose-derived Stem Cells Proliferation and in vivo Vascularization

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Spencer Brown ◽  
Francis Caputo ◽  
Marc Fromer ◽  
Ping Zhang ◽  
Shauhoa Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Endothelial Cell ◽  
High Glucose ◽  
Umbilical Vein ◽  
Adipose Derived Stem Cells ◽  
Diabetic Mice ◽  
Human Umbilical Vein

Background: Diabetes type 1 and 2 cause hyperglycemia and result in endothelial dysfunction with endothelial vessel and poor wound healing. Adipose-derived stem cells (ASCs), progenitor cells in wound healing, show decreased function under hyperglycemic conditions in vitro and in vivo . We hypothesized that exposing ASCs in the presence of high glucose with the human umbilical vein endothelial cell (HUVEC) secretome will reverse the deleterious effects of glucose on ASCs and subsequently enhance angiogenesis and wound healing. Methods: Human umbilical vein endothelial cells (HUVEC) were treated with glucose (30mM) and the conditioned media (CM) were collected every 3 days. ASCs were then co-cultured with EC/CM for 2 weeks. To produce thermal denaturation of protein, EC/CM was heated at 95 0 C for 30 mins. Cell activity, proliferation, and endothelial-like properties of ASCs were determined by MTT assays, growth curves, and real-time RT-PCR, respectively. EC/CM treated ASC were injected into a normal or diabetic murine left thigh muscle at three different points with hindlimb ischemia. After 4 weeks injection, animals were sacrificed. H & E and double immunostaining for CD31 and anti-human nuclei were used to determine if the ASCs primed with EC/CM underwent neovascularization. Results: In fact, ASCs increased in proliferation when co-cultured with HUVEC/CM (1.4 fold) when compared with controls. This promoting effect was lost in heated HUVEC/CM, indicating that the active molecules are of protein origin. After 10 days stimulated with EC/CM an increase in mRNA expression levels of EC markers were also observed in high glucose (30mM) EC/CM environment including CD31 (2-fold), vWF (1.1-fold), and eNOS (3.2-fold) when compared to ASCs cultured in M199. H & E and immunohistochemical staining results showed elevated vessel density and CD31 + cell levels in HUVEC-primed ASC injection sites of diabetic mice when compared with the control animals. Conclusions: HUVEC secrete protein factors that increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to improved angiogenesis.

scholarly journals Stamina-Enhancing Effects of Human Adipose-Derived Stem Cells

2021 ◽  
Vol 30 ◽  
pp. 096368972110354
Author(s):  
Eun-Jung Yoon ◽  
Hye Rim Seong ◽  
Jangbeen Kyung ◽  
Dajeong Kim ◽  
Sangryong Park ◽  
...  
Keyword(s):  
Physical Activity ◽  
Stem Cells ◽  
Locomotor Activity ◽  
Tissue Injury ◽  
Male Rats ◽  
Adipose Derived Stem Cells ◽  
Sprague Dawley ◽  
Serum Triglycerides

Stamina-enhancing effects of human adipose derived stem cells (hADSCs) were investigated in young Sprague-Dawley rats. Ten-day-old male rats were transplanted intravenously (IV) or intracerebroventricularly (ICV) with hADSCs (1 × 106 cells/rat), and physical activity was measured by locomotor activity and rota-rod performance at post-natal day (PND) 14, 20, 30, and 40, as well as a forced swimming test at PND 41. hADSCs injection increased the moving time in locomotor activity, the latency in rota-rod performance, and the maximum swimming time. For the improvement of physical activity, ICV transplantation was superior to IV injection. In biochemical analyses, ICV transplantation of hADSCs markedly reduced serum creatine phosphokinase, lactate dehydrogenase, alanine transaminase, and muscular lipid peroxidation, the markers for muscular and hepatic injuries, despite the reduction in muscular glycogen and serum triglycerides as energy sources. Notably, hADSCs secreted brain-derived neurotrophic factor (BDNF) and nerve growth factor in vitro, and increased the level of BDNF in the brain and muscles in vivo. The results indicate that hADSCs enhance physical activity including stamina not only by attenuating tissue injury, but also by strengthening the muscles via production of BDNF.

scholarly journals Stem cells and exosomes: promising candidates for necrotizing enterocolitis therapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ruijie Zeng ◽  
Jinghua Wang ◽  
Zewei Zhuo ◽  
Yujun Luo ◽  
Weihong Sha ◽  
...  
Keyword(s):  
Stem Cells ◽  
Necrotizing Enterocolitis ◽  
Therapeutic Effects ◽  
Beneficial Effects ◽  
Pediatric Diseases ◽  
Gastrointestinal Conditions ◽  
Different Types ◽  
Novel Therapeutic Strategies

AbstractNecrotizing enterocolitis (NEC) is a devastating disease predominately affecting neonates. Despite therapeutic advances, NEC remains the leading cause of mortality due to gastrointestinal conditions in neonates. Stem cells have been exploited in various diseases, and the application of different types of stem cells in the NEC therapy is explored in the past decade. However, stem cell transplantation possesses several deficiencies, and exosomes are considered potent alternatives. Exosomes, especially those derived from stem cells and breast milk, demonstrate beneficial effects for NEC both in vivo and in vitro and emerge as promising options for clinical practice. In this review, the function and therapeutic effects of stem cells and exosomes for NEC are investigated and summarized, which provide insights for the development and application of novel therapeutic strategies in pediatric diseases. Further elucidation of mechanisms, improvement in preparation, bioengineering, and administration, as well as rigorous clinical trials are warranted.

scholarly journals High glucose induces early senescence in adipose-derived stem cells by accelerating p16 and mTOR

2019 ◽  
Vol 6 (6) ◽  
pp. 3213-3221
Author(s):  
Hieu Liem Pham ◽  
Phuc Van Pham
Keyword(s):  
Stem Cells ◽  
High Glucose ◽  
Glucose Concentration ◽  
Culture Medium ◽  
Diabetic Patients ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Normal Glucose

Introduction: The senescence of stem cells is the primary reason that causes aging of stem cell-containing tissues. Some hypotheses have suggested that high glucose concentration in diabetic patients is the main factor that causes senescence of cells in those patients. This study aimed to evaluate the effects of high glucose concentrations on the senescence of adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated and expanded from human adipose tissues. They were characterized and confirmed as mesenchymal stem cells (MSCs) by expression of surface markers, their shape, and in vitro differentiation potential. They were then cultured in 3 different media- that contained 17.5 mM, 35 mM, or 55 mM of D-glucose. The senescent status of ADSCs was recorded by the expression of the enzyme beta-galactosidase, cell proliferation, and doubling time. Real-time RT-PCR was used to evaluate the expression of p16, p21, p53 and mTOR. Results: The results showed that high glucose concentrations (35 mM and 55 mM) in the culture medium induced senescence of human ADSCs. The ADSCs could progress to the senescent status quicker than those cultured in the lower glucose-containing medium (17.5 mM). The senescent state was related to the up-regulation of p16 and mTOR genes. Conclusion: These results suggest that high glucose in culture medium can trigger the expression of p16 and mTOR genes which cause early senescence in ADSCs. Therefore, ADSCs should be cultured in low glucose culture medium, or normal glucose concentration, to extend their life in vitro as well as in vivo.  

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