Resumen

RODRIGUES-PINTO, Ricardo; OLIVEIRA, António; RICHARDSON, Stephen M.  y  HOYLAND, Judith A.. Isolamento e caracterização das células da notocorda humana: Implicações para o desenvolvimento de terapias celulares para a doença degenerativa discal. Rev. Port. Ortop. Traum. [online]. 2013, vol.21, n.4, pp.443-472. ISSN 1646-2122.

Introduction: For the development of cell-based therapies targeting intervertebral disc (IVD) degeneration a thorough understanding of its cells and the molecular pathways leading to their differentiation is required. The embryonic, foetal and juvenile nucleus pulposus (NP) is populated by large vacuolated notochordal cells that with skeletal maturity become replaced by smaller chondrocyte-like cells. Since animal and in vitro studies have shown that notochordal cells display protective and anabolic roles in the IVD, their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of these cells and their regulatory pathways may help to identify factors involved in IVD homeostasis and the development of novel cell-based therapies targeting disc degeneration. The study of human notochordal cells has however been hindered by ethical, logistical and technical restrictions in obtaining suitable samples, which constitutes a major limitation to the field. This study was conducted with the objective of isolating notochordal cells (precursors of the NP) from their adjacent sclerotomal tissues (precursors of the annulus fibrosus (AF) and vertebrae) from human embryonic and foetal spines and to identify their phenotype and potential factors involved in their function in the IVD. Methods: Human embryonic and foetal spines (3.5-12 weeks post-conception) were dissected and characterised morphologically to confirm their suitability as sources of notochordal cells. Then, and since there was no visible demarcation between the NP, AF and vertebral regions, a strategy involving the identification of a notochord-specific marker to label and sort notochordal cells was devised. To identify a notochordspecific marker, a literature search was performed followed by the validation of identified putative markers in the human foetal spine using immunohistochemistry. Subsequently, and as the identified marker had an intracellular location, a novel methodology was developed to allow for the extraction of high quality RNA from limited numbers of fixed, permeabilised, labelled with a fluorescent antibody and sorted cells. Using the optimised methodology, RNA was extracted from notochordal and sclerotomal cells. Separation accuracy was validated using real time quantitative polymerase chain reaction (qRT-PCR) for known notochordal markers. Microarrays were then used to identify differentially expressed genes between notochordal and sclerotomal cells and interactive pathway analysis (IPA) was used to identify notochordal cell regulators. Results: The human foetal spine was validated as a suitable source of notochordal cells. The literature search identified 16 putative markers and the validation of a panel of these identified KRT8, KRT18 and KRT19 as human notochord-specific markers. A methodology involving cell fixation and permeabilisation using 95% ethanol/ 5% acetic acid, followed by incubation with a directly conjugated KRT18 antibody and separation by fluorescence activated cell sorting was found to have no detrimental effect on the RNA quality. This method was therefore used to separate enzymatically obtained human foetal notochordal cells (NP precursors and KRT18-positive) from sclerotomal cells (AF and vertebrae precursors and KRT18-negative). qRT-PCR analysis validated the cell separation methodology by confirming that notochordal cells had higher differential expression of the notochordal markers KRT18, KRT19, brachyury, Galectin 3, CTGF and FOXA2. Array analysis identified 782 upregulated and 678 downregulated genes. IPA analysis identified 30 upstream regulators of notochordal cell genes. Of these, hepatocyte growth factor (HGF) was the top growth factor and was predicted to be activating a number of relevant notochordal genes. Discussion: This study isolated, for the first time, human notochordal cells. To do so, a complex labelling methodology was devised that is applicable to different areas of research. Microarray analysis identified a list of notochordal cell markers, some of which may be important in notochordal cell function within the IVD. Importantly, HGF was identified as an upstream regulator of notochordal cells that may be involved in some of the biological functions attributed to them.

Palabras clave : Intervertebral disc; nucleus pulposus; disc degeneration; notochordal cells; phenotype; microarrays; regeneration; cellular therapies.

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