Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species.

STUDY DESIGN: Descriptive experimental study in 4 different mammals. OBJECTIVE: To investigate cell proliferation/regeneration and localize stem cells/progenitor cells within the intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA: Disc degeneration (DD) is believed to play a major role in patients with chronic lumbar pain. Lately, biologic treatment options for DD have gained increasing interest. Normal regeneration processes within the IVD and have previously been sparsely described and therefore it is of great interest to increase the knowledge about these processes. Methods. Detection of cell proliferations zones and label-retaining cells were done by in vivo 5-bromo-2-deoxyuridine (BrdU) labeling in 18 rabbits, killed after 4, 6, 10, 14, 28, or 56 days. Results were visualized with immunohistochemistry and fluorescence/confocal microscopy. Localization of progenitor cell were further investigated by immunohistochemistry using antibodies towards Notch1, Delta4, Jagged1, C-KIT, KI67, and Stro-1 in normal IVD from rabbits (n = 3), rats (n = 2), minipigs (n = 2), and in human degenerated IVD (n = 4). Further, flowcytometry analysis using progenitor markers were performed on additional human IVD cells (n = 3). RESULTS: BrdU positive cells were found in comparable numbers at early and late time points in most regions of the anulus fibrosus (AF) and nucleus pulposus demonstrating slow ongoing cell proliferation. In the AF border to ligament zone (AFo) and the perichondrium region (P) a stem cell niche-like pattern was determined (a high number of BrdU positive cells at early time points vs. only a few label retaining cells at later time points). In normal and DD tissue from the 4 investigated species progenitor cell markers were detected. Conclusion. The IVD is a tissue with ongoing slow cell proliferation both in the AF and the nucleus pulposus. The stem cell niche pattern detected in AFo and P can be suggested to play a role for IVD morphology and function. These findings may be of importance for the development of biologic treatment strategies.

Identification of a stem cell niche in the zone of Ranvier within the knee joint.

A superficial lesion of the articular cartilage does not spontaneously self-repair and has been suggested to be partly due to lack of progenitor cells within the joint that can reach the site of injury. To study whether progenitor cells are present within the joint, 3-month-old New Zealand white rabbits were exposed to bromodeoxyuridine (BrdU) for 12 consecutive days and were then sacrificed 4, 6, 10, 14, 28 and 56 days after the first BrdU administration. Presence of BrdU and localization of progenitor markers were detected using immunohistochemistry. After 10 days of BrdU exposure, BrdU-positive cells, i.e. proliferating cells, were abundantly detected in the epiphyseal plate, the perichondrial groove of Ranvier, and in all zones of the articular cartilage. After a wash-out period, BrdU-positive cells were still present, i.e. those considered to be progenitor cells, in these regions of the knee except for the proliferative zone of the epiphyseal plate. Cells in the perichondrial groove of Ranvier were further positive for several markers associated with progenitor cells and stem cell niches, including Stro-1, Jagged1, and BMPr1a. Our results demonstrate that a small population of progenitor cells is present in the perichondrial groove of Ranvier as well as within the articular cartilage in the knee. The perichondrial groove of Ranvier also demonstrates the properties of a stem cell niche.

The helix-loop-helix transcription factor Id1 is highly expressed in psoriatic involved skin.

The helix-loop-helix transcription factor Id1 (inhibitor of differentiation/inhibitor of DNA binding) functions as an inhibitor of differentiation. We have examined Id1 gene expression in cultured keratinocytes in punch biopsies from psoriatic involved and uninvolved skin, and in skin specimens from normal individuals. Id1 mRNA expression was measured with an RNase protection assay and with Northern blot. Id1 immunoreactivity was determined in skin biopsies by immunofluorescence using a polyclonal antibody directed against the Id1 protein. In cultured keratinocytes, the expression of Id1 mRNA was strongest in small cells with high proliferative potential, whereas in large cells, which are terminally differentiated, the expression was low. Expression of the Id1 mRNA in psoriatic involved skin (n = 9) was significantly elevated compared to uninvolved skin from the same patient (n = 5) and to skin from normal controls (n = 9). Id1 immunoreactivity was intranuclear throughout all the layers in psoriatic involved epidermis, except in the stratum corneum, while no immunoreactivity was detected in uninvolved epidermis. In normal controls, cytoplasmatic Id1 immunoreactivity was detected in the basal layer in epidermis obtained from newborns, while no immunoreactivity was detected in epidermis obtained from the adults in the control group. We conclude that Id1 is expressed in cells with high proliferative potential, and is downregulated in cells that undergo terminal differentiation. Along with the overexpression of the Id1 gene in psoriatic involved skin, these observations suggest that Id1 is involved in the process of differentiation of keratinocytes seen in normal skin and that the Id1 pathway is activated in psoriasis.

Identification of AHNAK as a novel autoantigen in systemic lupus erythematosus.

To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis. One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways. Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors. Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations. This is the first report that AHNAK can be targeted by the immune system in autoimmune disease.