Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro.
METHODS
We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells.


OBJECTIVE
To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager.


BACKGROUND
Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors.


METHODS
Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study.


RESULTS
Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% +/- 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 +/- 507.1 microm, velocity: 0.09 +/- 0.01 microm/min); giant cells (area: 12678 +/- 1637.0 microm, velocity: 0.08 +/- 0.01 microm/min) which grew rapidly without cell division; polygonal cells (area: 3053 +/- 751.2 microm, 0.14 +/- 0.01 microm/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 +/- 235.6 microm, 0.19 +/- 0.01 microm/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo.


CONCLUSIONS
These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.
Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Joo Han
JH
Kim
Bridget M
BM
Deasy
Hyoung Yeon
HY
Seo
Rebecca K
RK
Studer
Nam V
NV
Vo
Helga I
HI
Georgescu
Gwendolyn A
GA
Sowa
James D
JD
Kang
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D048430:Cell Shape / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D003094:Collagen / Q000235:genetics / Q000378:metabolism; D003952:Diagnostic Imaging / Q000379:methods*; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009672:Notochord / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010641:Phenotype; D011509:Proteoglycans / Q000096:biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000378:metabolism
D053674:Aggrecans; D011509:Proteoglycans; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor; D003094:Collagen