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New research underscores potential for stem cell use in disc regeneration
Culture conditions affect the ability of mesenchymal stem cells to differentiate into intervertebral disc cells.
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VANCOUVER, Canada — Techniques that may employ mesenchymal stem cells to regenerate cells of degenerated human intervertebral discs showed that microenvironment plays an important role in cell proliferation, as does the cells’ ability to produce extracellular matrix.
Results of three laboratory studies and one using a rabbit model that investigated these areas were presented here during the International Society for the Study of the Lumbar Spine 30th Annual Meeting.
The concept of rescuing degenerative discs by injecting them with cultured mesenchymal stem cells (MSC) is not new, but more information is needed about the best MSC sources, culture conditions that support cell differentiation, and techniques that yield the greatest number of cells.
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“Mesenchymal stem cells have the potential as an ultimate alternative in cell transplantation therapy for degenerative disc disease,” said Daisuke Sakai, MD, who presented results of a study that looked at autologous transplantation of MSCs into artificially degenerated discs of New Zealand white rabbits.
Researchers at Tokai University School of Medicine in Kanagawa, Japan, found that within the disc environment, transplanted MSCs differentiated into disc-like cells, survived and proliferated, preserving disc structure.
Disc height 26 weeks after degeneration was 90 ±8% in the transplanted group compared to 67 ±8% in controls, Sakai told Orthopaedics Today in an e-mail interview. MSC transplantation restored synthesis of a proteoglycan-rich matrix.
“Our study has implicated the potential of MSCs to differentiate into intervertebral disc cells, which provides new information in MSC research,” the researchers wrote in an abstract of their paper.
Disc cells and MSC interaction
A study conducted by investigators at Johns Hopkins University School of Medicine in Baltimore, U.S.A, investigated whether bone marrow-derived MSCs could stimulate the anulus fibrosus (AF) and nucleus pulposus (NP) cells they might eventually interact with during clinical application, lead investigator Catherine Le Visage, PhD, said in an e-mail interview.
Human NP and AF cells isolated from adult degenerative discs cultured in a commercially available MSC medium yielded higher glycosaminoglycan (GAG) content, which increased over time, compared to controls of NP and AF cells, chondrocytes and fibroblasts cultured in DMEM/F12 medium. For example, between the first and third week, GAG content in NP cells in MSC medium increased from 1.8 to 4.3 µg/pellet, compared to increases from 1.1 to 1.4 µg/pellet in NP cell controls.
Subsequent MSC co-culture experiments yielded AF cells with consistently higher GAG content values than predicted. “Co-cultured disc cells and MSC could aggregate and produce an extracellular matrix, and there is upregulation of the GAG content between the cells,” said Eiji Hanaoka, MD, PhD, who presented the findings.
“This preliminary study strongly suggests that degenerative disc cells may be stimulated in vivo by implantation of autologous MSCs to restore some of the disc properties,” Le Visage said.
NP phenotype
A study of NP cell lineage conducted in the department of orthopaedic surgery at Thomas Jefferson University in Philadelphia, U.S.A demonstrated that given the proper microconditions, MSCs can express a phenotype like that of NP cells.
Researchers placed MSCs in an alginate-capsulated environment and
cultured them for seven days to determine the effect of normoxia and hypoxia,
with high and low osmolarity. Semiquantitative real-time PCR analysis showed
that MSCs expressed transcripts for such hypoxia-sensitive genes as MMP-2,
GLUT-3 and HIF-2
. Also
expressed were collagen 2 and 11, aggrecan, decorin and biglycan, as well as
proteins for MMP-2 and HIF-2 based on Western blot analysis.
Cell clumping commenced in normoxia, but “when you look at hypoxia, you see even healthier cells with more cell clumping in the normal osmolarity,” said presenter Todd J. Albert, MD. “Hypoxic conditions in combination with the alginate culture and the growth factor that we chose, which was TGF-ß, were really optimal for achieving an NP-like phenotype.”
Transfected stem cells
Members of the Injury, Repair and Rehabilitation Research Group at the University of Manchester, England, cultured adult human MSCs in monolayer and in chondrogenic media (ie, pellets or alginate beads), inducing them to differentiate along a chondrocytic NP-like phenotype.
MSCs cultured in alginate and pellet culture had the most chondrogenic characteristics, forming an extracellular matrix similar to that seen in vivo. When the MSCs were cultured in these media, however, “Cells were not able to divide and were not easily accessible,” said Stephen M. Richardson, PhD, who presented the results.
MSCs grown in monolayer in basic media transfected with SOX-9 differentiated and expressed type II collagen, aggrecan and SOX-9, but not type I collagen. This is similar to that seen in vivo and in pellet or alginate cultures, but the cells continued to divide.
“Therefore SOX-9 transfected stem cells in monolayer may provide a useful source of cells for differentiating into normal NP cells,” Richardson said.
Differentiation improved and there was high-level matrix production using TGF-ß1 in combination with SOX-9, he added.
Assess new technology
During the discussion period, session moderator Björn L. Rydevik, MD, PhD, of Gothenburg, Sweden, called this “a very challenging line of research” and provided perspective for future research into disc regeneration using MSCs.
“It’s wise to perhaps move forward in a stepwise manner, to gradually evaluate this new technology. The total processes have to be constantly reassessed,” he said.
For your information:
- Sakai D, Mochida J, Yamamoto Y, et al. Autologous transplantation of mesenchymal stem cells for disc repair. #24.
- Le Visage C, Tateno K, Sieber A, et al. Interaction of human mesenchymal stem cells with disc cells: changes in biosynthesis of extracellular matrix. #25.
- Risbud M, Izzo M, Adams C, et al. Mesenchymal stem cells respond to their microenvironment in vitro to assume nucleus pulposus-like phenotype. #26.
- Richardson S, Le Maitre C, Russell A, et al. Human bone marrow mesenchymal stromal cells as a source of chondrocytes for treatment of intervertebral disc degeneration. #27. Presented at the International Society for the Study of the Lumbar Spine 30th Annual Meeting. May 13-17, 2003. Vancouver, Canada