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Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

ABSTRACT: Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPAR?2. ADSCs could differentiate into neural cells, stained positive to ?-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

SUBMITTER: Kumar A

PROVIDER: S-EPMC6938680 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

Kumar Ajay A Xu Yi Y Yang Enzhi E Wang Yiwen Y Du Yiqin Y

Experimental eye research 20191023

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-ye ...[more]

PMID: 31655040

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Project description:BackgroundPerivascular adipose-derived stem cells (PVASCs) can contribute to vascular remodeling, which are also capable of differentiating into multiple cell lineages. The present study aims to investigate the mechanism of PVASC differentiation toward smooth muscle cells (SMCs) and endothelial cells (ECs) as well as its function in neointimal hyperplasia.MethodsSingle-cell sequencing and bulk mRNA sequencing were applied for searching key genes in PVASC regarding its role in vascular remodeling. PVASCs were induced to differentiate toward SMCs and ECs in vitro, which was quantitatively evaluated using immunofluorescence, quantitative real-time PCR (QPCR), and Western blot. Lentivirus transfections were performed in PVASCs to knock down or overexpress TBX20. In vivo, PVASCs transfected with lentivirus were transplanted around the guidewire injured femoral artery. Hematoxylin-eosin (H&E) staining was performed to examine their effects on neointimal hyperplasia.ResultsBulk mRNA sequencing and single-cell sequencing revealed a unique expression of TBX20 in PVASCs. TBX20 expression markedly decreased during smooth muscle differentiation while it increased during endothelial differentiation of PVASCs. TBX20 knockdown resulted in the upregulation of SMC-specific marker expression and activated Smad2/3 signaling, while inhibiting endothelial differentiation. In contrast, TBX20 overexpression repressed the differentiation of PVASCs toward smooth muscle cells but promoted endothelial differentiation in vitro. Transplantation of PVASCs transfected with TBX20 overexpression lentivirus inhibited neointimal hyperplasia in a murine femoral artery guidewire injury model. On the contrary, neointimal hyperplasia significantly increased in the TBX20 knockdown group.ConclusionA subpopulation of PVASCs uniquely expressed TBX20. TBX20 could regulate SMC and EC differentiation of PVASCs in vitro. Transplantation of PVASCs after vascular injury suggested that PVASCs participated in neointimal hyperplasia via TBX20.

Project description:Human adipose tissue has been recently recognized as a potential source of stem cells for regenerative medicine applications, including bone tissue engineering (TE). Despite the gathered knowledge regarding the differentiation potential of human adipose tissue-derived stem cells (hASCs), in what concerns the endothelial lineage many uncertainties are still present. The existence of a cell subpopulation within the human adipose tissue that expresses a SSEA-4 marker, usually associated to pluripotency, raises expectations on the differentiation capacity of these cells (SSEA-4(+)hASCs). In the present study, the endothelial and osteogenic differentiation potential of the SSEA-4(+)hASCs was analyzed, aiming at proposing a single-cell source/subpopulation for the development of vascularized bone TE constructs. SSEA-4(+)hASCs were isolated using immunomagnetic sorting and cultured either in α-MEM, in EGM-2 MV (endothelial growth medium), or in osteogenic medium. SSEA-4(+)hASCs cultured in EGM-2 MV formed endothelial cell-like colonies characterized by a cobblestone morphology and expression of CD31, CD34, CD105, and von Willebrand factor as determined by quantitative reverse transcriptase (RT)-polymerase chain reaction, immunofluorescence, and flow cytometry. The endothelial phenotype was also confirmed by their ability to incorporate acetylated low-density lipoprotein and to form capillary-like structures when seeded on Matrigel. SSEA-4(+)hASCs cultured in α-MEM displayed fibroblastic-like morphology and exhibited a mesenchymal surface marker profile (>90% CD90(+)/CD73(+)/CD105(+)). After culture in osteogenic conditions, an overexpression of osteogenic-related markers (osteopontin and osteocalcin) was observed both at molecular and protein levels. Matrix mineralization detected by Alizarin Red staining confirmed SSEA-4(+)hASCs osteogenic differentiation. Herein, we demonstrate that from a single-cell source, human adipose tissue, and by selecting the appropriate subpopulation it is possible to obtain microvascular-like endothelial cells and osteoblasts, the most relevant cell types for the creation of vascularized bone tissue-engineered constructs.

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Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

Publications

Fidelity of long-term cryopreserved adipose-derived stem cells for differentiation into cells of ocular and other lineages.

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