Human umbilical cord blood-derived mesenchymal stem cells in the cultured rabbit intervertebral disc: a novel cell source for disc repair

Abstract

Objective: Back pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture.

Design: This study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant tracer specific for live cells. Human type II collagen gene expression (from the graft) was assessed by polymerase chain reaction.

Results: We have shown that hUCB-MSCs cultured in micromass are stained blue with Alcian blue dye, which suggests that proteoglycan-rich extracellular matrix is produced. In the cultured rabbit IVD explants, hUCB-MSCs survived for at least 4 wks and expressed the human type II collagen gene, suggesting that the injected hUCB-MSCs are differentiating into a chondrocyte-like lineage.

Conclusions: This study demonstrates the abiity of hUBC-MSCs to survive and assume a chondrocyte-like phenotype when injected into the rabbit IVD. These data support the potential for hUBC-MSCs as a cell source for disc repair. Further measures of the host response to the injection and studies in animal models are needed before trials in humans.

FIGURE 1

Human umbilical cord blood–derived mesenchymal stem cells cultured as micropellets stained with Alcian blue for proteoglycans. A and B, micropellets stained with Alcian blue and counterstained with hematoxylin and eosin (H&E) and examined by stereomicroscopy. C and D, paraffin sections of the micropellets stained with H&E. A and C, micropellets cultured without transforming growth factor (TGF)-β_3. B and D, micropellets cultured in the presence of TGF-β_3. Panels A′-D′ are high-magnification images of boxed areas in panels A–D, respectively.

FIGURE 2

Human umbilical cord blood–derived mesenchymal stem cells cultured as micropellets immunostained for type II collagen. A and A′, pellet cultured with transforming growth factor (TGF)-β. B and B′, pellet cultured without TGF-β. A and B, nonimmune rabbit immunoglobulin G as negative control; A′ and B′, immunostained with anti–type II collagen primary antibody.

FIGURE 3

Human umbilical cord blood–derived mesenchymal stem cells (hUCB-MSCs) persisted for up to 4 wks after injection into cultured rabbit intervertebral disc (IVD) explants. A, rabbit IVDs injected with infrared dye–labeled hUCB-MSCs were cultured and scanned weekly by a LI-COR scanner. The injected cells were detected in the 800-nm-wavelength channel and is represented in green. The rabbit disc contour was detected in the 700-nm-wavelength channel and is represented in red. Colocalization of the injected cells (green) and disc contour (red) was represented in yellow. B, integrated intensity of the fluorescence in the 800-nm-wavelength in the IVD. Error bar: standard error of the mean. *P < 0.05.

FIGURE 4

Human umbilical cord blood–derived mesenchymal stem cells (hUCB-MSCs) were viable 4 wks after injection into cultured rabbit intervertebral disc explants. Nucleus pulposus (NP) tissues were scooped out and examined by confocal fluorescent microscopy. A, hUCB-MSCs stained with DiI were detected by their red fluorescence. B, live cells stained with CellTracker Green were detected by their green fluorescence. C, the overlay of panels A and B illustrating the integration of the transplanted cells (orange) into the viable native NP tissue (green). Arrows in all three panels point to injected cells.

FIGURE 5

hCOL2 gene expression by RT-PCR 4 wks after hUCB-MSC injection into the RD. A, polymerase chain reaction products separated on agarose gel. Lane 1, hUCB-MSC cultured in monolayer; lane 2, RD; lane 3, rabbit disc injected with hUCB-MSC (RD + MSC). B, ratio of hCOL2 to hGAPDH density. hCOL2 indicates human type II collagen; RT, PCR, reverse transcription polymerase chain reaction; hUCB-MSC, human umbilical cord blood–derived mesenchymal stem cell; RD, rabbit disc; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; rGAPDH, rabbit glyceraldehyde-3-phosphate dehydrogenase.