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Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened ...

Christian Medical College

Resident articular stem cells isolated using a migratory assay called Migratory Chondroprogenitors (MCPs) have emerged as a promising cellular therapeutic for the treatment of cartilage pathologies. In-vivo studies using MCPs report their superiority over bone-marrow mesenchymal stem cells and chondrocytes for treating chondral defects. However, there is no consensus on their isolation protocol. This study aimed to compare four reported isolation methods of MCPs and identify the optimal and feasible protocol for future translational work.

Comparison of methods for the isolation and culture of Migratory chondroprogenitors from Human articular cartilage.

Obtaining regeneration-competent cells and generating high-quality neocartilage are still challenges in articular cartilage tissue engineering. Although chondroprogenitor cells are a resident subpopulation of native cartilage and possess a high capacity for proliferation and cartilage formation, their potential for regenerative medicine has not been adequately explored. Fetal cartilage, another potential source with greater cellularity and a higher cell-matrix ratio than adult tissue, has been evaluated for sourcing cells to treat articular disorders. This study aimed to compare cartilage resident cells, namely chondrocytes, fibronectin adhesion assay-derived chondroprogenitors (FAA-CPCs) and migratory chondroprogenitors (MCPs) isolated from fetal and adult cartilage, to evaluate differences in their biological properties and their potential for cartilage repair. Following informed consent, three human fetal and three adult osteoarthritic knee joints were used to harvest the cartilage samples, from which the three cell types a) chondrocytes, b) FAA-CPCs, and MCPs were isolated. Assessment parameters consisted of flow cytometry analysis for percentage expression of cell surface markers, population doubling time and cell cycle analyses, qRT-PCR for markers of chondrogenesis and hypertrophy, trilineage differentiation potential and biochemical analysis of differentiated chondrogenic pellets for total GAG/DNA content. Compared to their adult counterparts, fetal cartilage-derived cells displayed significantly lower CD106 and higher levels of CD146 expression, indicative of their superior chondrogenic capacity. Moreover, all fetal groups demonstrated significantly higher levels of GAG/DNA ratio with enhanced uptake of collagen type 2 and GAG stains on histology. It was also noted that fetal FAA CPCs had a greater proliferative ability with significantly higher levels of the primary transcription factor SOX-9. Fetal chondrocytes and chondroprogenitors displayed a superior propensity for chondrogenesis when compared to their adult counterparts. To understand their therapeutic potential and provide an important solution to long-standing challenges in cartilage tissue engineering, focused research into its regenerative properties using in-vivo models is warranted.

Human fetal cartilage-derived chondrocytes and chondroprogenitors display a greater commitment to chondrogenesis than adult cartilage resident cells.

Cell-based therapeutics for articular cartilage repair primarily employed bone marrow-derived mesenchymal stem cells and chondrocytes. Research to overcome their limitation of formation of a functionally poor fibro-hyaline type of repair tissue led to the discovery of chondroprogenitors (CPCs), cartilage resident stem cells. These cells isolated by adhesion assay using fibronectin (FAA-CPs) and migration of progenitors from explants (MCPs) display higher chondrogenic and lower terminal differentiation potential. During in-vitro culture, chondrocytes tend to de-differentiate and acquire characteristics similar to stem cells, thus making it challenging to distinguish them from other cell groups. Ghrelin, a cytoplasmic growth hormone secretagogue, has been proposed to play a vital role in chondrogenesis, with reports of its higher expression in chondrocytes than BM-MSCs. The aim of this study was to compare the mRNA expression of Ghrelin between BM-MSCs, chondrocytes, FAA-CPs and MCP and the possibility of it serving as a distinguishing marker.

Evaluation of ghrelin as a distinguishing marker for human articular cartilage-derived chondrocytes and chondroprogenitors.

The recent discovery of progenitors based on their differential fibronectin-adhesion (FAA-CPs) and migratory-based (MCPs) assay has evoked interest due to their superiority in terms of their efficient chondrogenesis and reduced hypertrophic propensity. This study aims to isolate and enrich three articular cartilage subsets, chondrocytes, FAA-CPs, and MCPs, and compare their undifferentiated and chondrogenic differentiated status, using in-vitro phenotypical characterization in correlation with ultrastructural analysis using Transmission Electron Microscopy (TEM). Following informed consent, cartilage shavings were procured from a non-diseased human ankle joint and cultured to obtain the three subsets. Chondrocytes exhibited higher CD106 and lower CD49b and CD146 levels. Following chondrogenic differentiation, corroborative results were seen, with the MCP group showing the highest GAG/DNA ratio levels and uptake of extracellular matrix stain as compared to the FAA-CP group. TEM analysis of the chondrocytes revealed the presence of more autolytic cells with disintegrated cytoplasm and plasma membrane. The differentiated FAA-CPs and MCPs displayed higher collagen and rough endoplasmic reticulum. The results presented in this study provide novel information on the ultrastructural characteristics of cartilage resident cells, with the chondrocyte group displaying features of terminal differentiation. Both progenitor subtypes showed superiority in varied contexts, with greater collagen fibrils and greater GAG content in MCPs. The display of preferential and differentiation traits sheds insight on the necessity to enrich progenitors and coculturing them with the general pool of constituent cells to combine their advantages and reduce their drawbacks to achieve a regenerative tissue displaying genuine hyaline-like repair while limiting their terminal differentiation.

An ultrastructural report of human articular cartilage resident cells in correlation with their phenotypic characteristics.

Despite the success of Tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML), leukemic stem cells (LSCs) persist, contributing to relapse and resistance. CML Mesenchymal Stromal Cells (MSCs) help in LSC maintenance and protection from TKIs. However, the limited passage and self-differentiation abilities of primary CML MSCs hinder extensive research. To overcome this, we generated and characterized an immortalised CML patient-derived MSC (iCML MSC) line and assessed its role in LSC maintenance. We also compared the immunophenotype and differentiation potential between primary CML MSCs at diagnosis, post-treatment, and with normal bone marrow MSCs. Notably, CML MSCs exhibited enhanced chondrogenic differentiation potential compared to normal MSCs. The iCML MSC line retained the trilineage differentiation potential and was genetically stable, enabling long-term investigations. Functional studies demonstrated that iCML MSCs protected CML CD34 cells from imatinib-induced apoptosis, recapitulating the bone marrow microenvironment-mediated resistance observed in patients. iCML MSC-conditioned media enabled CML CD34 and AML blast cells to proliferate rapidly, with no impact on healthy donor CD34 cells. Gene expression profiling revealed dysregulated genes associated with calcium metabolism in CML CD34 cells cocultured with iCML MSCs, providing insights into potential therapeutic targets. Further, cytokine profiling revealed that the primary CML MSC lines abundantly secreted 25 cytokines involved in immune regulation, supporting the hypothesis that CML MSCs create an immune modulatory microenvironment that promotes growth and protects against TKIs. Our study establishes the utility of iCML MSCs as a valuable model to investigate leukemic-stromal interactions and study candidate genes involved in mediating TKI resistance in CML LSCs.

Immortalised chronic myeloid leukemia (CML) derived mesenchymal stromal cells (MSCs) line retains the immunomodulatory and chemoprotective properties of CML patient-derived MSCs.

Chondroprogenitors, with enhanced chondrogenic potential, have emerged to be a promising alternative for cell-based therapy in cartilage repair. Platelet-rich plasma (PRP), widely used for intra-articular treatment, has a short half-life. Freeze-dried PRP (FD-PRP), with an extended half-life and retained growth factors, is gaining attention. This study compares the efficacy of Migratory Chondroprogenitors (MCPs) in gelled PRP and FD-PRP using in-vitro and ex-vivo models, assessing FD-PRP as a potential off-the-shelf option for effective cartilage repair.

Comparative assessment of chondral defect repair using migratory chondroprogenitors suspended in either gelled or freeze-dried platelet-rich plasma: An in vitro and ex vivo human osteochondral unit model study.

Ganesh Parasuraman

Centre for Stem Cell Research (A unit of InStem,
Bengaluru),
Centre for Stem Cell Research (A unit of InStem, Bengaluru)

Christian Medical College

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Ganesh Parasuraman

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Christian Medical College

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Centre for Stem Cell Research (A unit of InStem,
Bengaluru),
Centre for Stem Cell Research (A unit of InStem, Bengaluru)