Issue: July 2009
July 01, 2009
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Purified stem cell group shows greater levels of type II collagen vs. mixed group

Investigator says it is a step toward finding MSC subpopulations with chondrogenic potential.

Issue: July 2009
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MIAMI — Subpopulations of human mesenchymal stem cells (MSCs) may have varying potential for chondrogenic expression, according to a study conducted by Canadian researchers.

“Human MSC populations isolated from the bone marrow [are] heterogeneous,” Charles C. Secretan, MD, said during his presentation at the 8th World Congress of the International Cartilage Repair Society. “There appears to be subpopulations in human mesenchymal stem cells with functional and differential capabilities, and a CD44 purified mesenchymal stem cell population did show an enhanced ability to produce more type II collagen and aggrecan [compared to a mixed population].”

Different populations

ICRS

To determine if a purified subpopulation of MSCs would lead to a more uniform differentiation of cells into chondrocytes, Secretan and his colleagues investigated the CD44 cell surface receptor which is believed to play a role in cartilage matrix generation and homoeostasis.

Alex Vaisman, MD
Alex Vaisman

After culturing and isolating MSCs, the investigators used flow cytometry to detect the surface antigens in the population. They then created the following three groups of cells using a fluorescence-activated cell sorter:

  • a CD44-positive population;
  • a CD44-negative population; and
  • a mixed or native population.

They used real-time polymerase chain reaction to quantify and compare the type I collagen, type II collagen and aggrecan content in the stem cell-derived chondrocytes in each group.

Alkaline phosphatase
Alkaline phosphatase staining of mesenchymal stem cell (MSC) differentiated osteocytes.

Collagen II immunpfluorescence
Collagen II immunpfluorescence of MSC differentiated chondrocytes.

Images: Secretan CC

The investigators discovered significantly greater type II collagen expression in the CD44-positive population compared to the mixed and CD44-negative groups. The CD44-positive group also showed significantly greater aggrecan expression than the mixed population.

However, the investigators found no significant difference in the aggrecan expression between the CD44-positive and negative groups. All of the groups showed high levels of type I collagen, Secretan said.

He noted that the quantification performed in this work was relative and the statistically significant differences found may not be clinically significant.

FABP4 immunofluorescence
FABP4 immunofluorescence of MSC differentiated adipocytes.

Oil red O staining
Oil red O staining of MSC differentiated adipocytes.

Limitations

“This work demonstrated that MSC subpopulations with differing potential exist and with different potential for gene expression,” Secretan said. “It is a step toward identifying MSC subpopulations with enhanced chondrogenic potential. It also highlights the need for those working with these cells to standardize the isolation and culture conditions used.”

The session co-moderator, Alan J. Nixon, BVSc, MS, noted that different cell preparation conditions can optimize chondrogenesis. “Did you try different batches of fetal calf serum and are there differences between different doses?” he asked.

Secretan noted that the investigators used different batches in the laboratory, but one specific batch was used throughout the experiment. He had not compared the batches.

For more information:
  • Alan J. Nixon, BVSc, MS, is a professor of large animal surgery. He can be reached at Cornell University, College of Veterinary Medicine, Ithaca, NY 14853; 607-253-3224; e-mail: ajn1@cornell.edu.
  • Charles C. Secretan, MD, can be reached at the department of surgery, University of Alberta, Edmonton, Alberta, Canada. T6G 2B7; 780-407-6605; e-mail: secretan@ualberta.ca. They have no direct financial interest in any products or companies mentioned in this article.
Reference:
  • Secretan CC, Bater J, Bagnall KN, et al. Isolation of a subpopulation of human mesenchymal stem cells with enhanced chondrogenic potential. #9.2.5. Presented at the 8th World Congress of the International Cartilage Repair Society. May 23-26, 2009. Miami.