发表于Stem Cell Research Therapy的Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells一文中，来自美国华盛顿大学的Lori A. Setton及其研究团队开发了一种新的方法，用于在体外将人类诱导多能干细胞（hiPSCs）有效地分化成NP样细胞。这种分化策略源于研究者们对NP细胞的胚胎脊索细胞系的了解以及用于支持原代细胞体外健康NP细胞表型的策略。
Intervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro.
Methods
An NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry.
Results
Periods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LMα5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells.
Conclusions
The ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.
Stem Cell Research Therapy(https://stemcellres.biomedcentral.com/about, 4.963 - 2-year Impact Factor, 5.382 - 5-year Impact Factor) is the major forum for translational research into stem cell therapies. An international peer-reviewed journal, it publishes high-quality open access research articles with a special emphasis on basic, translational and clinical research into stem cell therapeutics and regenerative therapies, including animal models and clinical trials. The journal also provides reviews, viewpoints, commentaries, reports and methods.
