4. ISOLATION OF A SUBPOPULATION OF HUMAN MESENCHYMAL STEM CELLS WITH ENHANCED CHONDROGENIC POTENTIAL

Abstract

Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration.

Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes. Real time polymerase chain reaction was utilized to quantify and compare the essential markers, collagen II, collagen I and aggrecan, in the stem cell derived chondrocytes. The CD44 enriched and CD44 depleted populations were compared.

Results: The cells isolated possessed a cell morphology and surface antigen profile consistent with a MSC population. In addition, both experimental groups demonstrated multipotent ability. Real time PCR analysis of the chondrogenic cells demonstrated that the CD44 positive population expressed collagen II and aggrecan at a significantly higher level than the CD44 negative population.

Conclusion: To date no group has successfully identified a relationship between a MSC subpopulation and the multipotent progenitors responsible for generating cartilage. This work demonstrated that there are MSC sub-populations with different potential for chondrogenic expression and represents an important step towards identifying MSC subpopulations with enhanced cartilage formation potential.