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Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

Hamid AA, Idrus RB, Saim AB, Sathappan S, Chua KH

Clinics (Sao Paulo), 2012;67(2):99-106.
PMID: 22358233

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction.
MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.
RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.
CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.

Matched MeSH terms: Chondrocytes/cytology
Fulltext Cartilage regeneration by chondrogenic induced adult stem cells in osteoarthritic sheep model

Ude CC, Sulaiman SB, Min-Hwei N, Hui-Cheng C, Ahmad J, Yahaya NM, et al.

PLoS One, 2014;9(6):e98770.
PMID: 24911365 DOI: 10.1371/journal.pone.0098770

In this study, Adipose stem cells (ADSC) and bone marrow stem cells (BMSC), multipotent adult cells with the potentials for cartilage regenerations were induced to chondrogenic lineage and used for cartilage regenerations in surgically induced osteoarthritis in sheep model.

Matched MeSH terms: Chondrocytes/cytology
Stem cell genes are poorly expressed in chondrocytes from microtic cartilage

Ishak MF, Chua KH, Asma A, Saim L, Aminuddin BS, Ruszymah BH, et al.

Int J Pediatr Otorhinolaryngol, 2011 Jun;75(6):835-40.
PMID: 21543123 DOI: 10.1016/j.ijporl.2011.03.021

This study was aimed to see the difference between chondrocytes from normal cartilage compared to chondrocytes from microtic cartilage. Specific attentions were to characterize the growth of chondrocytes in terms of cell morphology, growth profile and RT-PCR analysis.

Matched MeSH terms: Chondrocytes/cytology*
Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy

Munirah S, Samsudin OC, Aminuddin BS, Ruszymah BH

Tissue Cell, 2010 Oct;42(5):282-92.
PMID: 20810142 DOI: 10.1016/j.tice.2010.07.002

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation.

Matched MeSH terms: Chondrocytes/cytology*
Isolation, Expansion, and Characterization of Wharton's Jelly-Derived Mesenchymal Stromal Cell: Method to Identify Functional Passages for Experiments

Aung SW, Abu Kasim NH, Ramasamy TS

Methods Mol Biol, 2019;2045:323-335.
PMID: 31201682 DOI: 10.1007/7651_2019_242

The therapeutic potential of human mesenchymal stromal stem cells (hMSCs) for cell-based therapeutic is greatly influenced by the in vitro culture condition including the culture conditions. Nevertheless, there are many technical challenges needed to be overcome prior to the clinical use including the quantity, quality, and heterogeneity of the cells. Therefore, it is necessary to develop a stem cell culture procedure or protocol for cell expansion in order to generate reproducible and high-quality cells in accordance with good manufacturing practice for clinical and therapeutic purposes. Here we assessed the MSCs characteristic of human Wharton's jelly mesenchymal stromal cells in in vitro culture according to the criteria established by the International Society for Cellular Therapy. Besides, the viability of the WJMSCs was determined in order to increase the confidence that the cells are employed to meet the therapeutic efficacy.

Matched MeSH terms: Chondrocytes/cytology
Fulltext Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes

Liau LL, Hassan MNFB, Tang YL, Ng MH, Law JX

Int J Mol Sci, 2021 Jan 28;22(3).
PMID: 33525349 DOI: 10.3390/ijms22031269

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient's quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes.

Matched MeSH terms: Chondrocytes/cytology
Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells

Tan KL, Chia WC, How CW, Tor YS, Show PL, Looi QHD, et al.

Mol Biotechnol, 2021 Sep;63(9):780-791.
PMID: 34061307 DOI: 10.1007/s12033-021-00339-2

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

Matched MeSH terms: Chondrocytes/cytology*
Ex vivo expanded SSEA-4+ human limbal stromal cells are multipotent and do not express other embryonic stem cell markers

Lim MN, Hussin NH, Othman A, Umapathy T, Baharuddin P, Jamal R, et al.

Mol Vis, 2012;18:1289-300.
PMID: 22665977

The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.

Matched MeSH terms: Chondrocytes/cytology
Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro

Choong PF, Mok PL, Cheong SK, Leong CF, Then KY

Cytotherapy, 2007;9(2):170-83.
PMID: 17453969

The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons.

Matched MeSH terms: Chondrocytes/cytology