Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells

Abstract

Background: 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.

Keywords: Cartilage; Chondrocyte; Degenerative disc disease; Directed differentiation; Human induced pluripotent stem cells; Intervertebral disc; Nucleus pulposus.

Fig. 1

Protocol design. Schematic diagram illustrating three steps for directed differentiation of undifferentiated hiPSCs into NP-like cells. E8 medium = Essential 8™ medium (STEMCELL Technologies, Seattle, WA, USA); hiPSC = human induced pluripotent stem cell; d = days; 6w = six-well; SHH = sonic hedgehog; T = brachyury; BMP4 = bone morphogenetic protein 4; FOXA2 = forkhead box protein A2; FGF2 = basic fibroblast growth factor; CD24 = cluster of differentiation 24 protein; BASP1 = brain abundant membrane attached signal protein 1; MIXL1 = paired-type homeobox transcription factor identified in human; CDX2 = member of the caudal-related homeobox transcription factor family; TGF = transforming growth factor; LMα5 = alpha-5 subunit of heterotrimeric laminin; NOG = noggin; NOTO = notochord homeobox

Fig. 2

Pluripotency for derived colonies confirmed via immunofluorescence. a Undifferentiated human iPSCs cultured in colonies on vitronectin-coated plates until induction of differentiation (scale bar = 50 μm). b Immunofluorescence showing DNA (green) and positive pluripotent marker expression (red) of TRA1–60, SOX2, OCT4, TRA1–81, and SSEA-4. Yellow indicates costaining of green nuclei and red pluripotent markers (scale bar = 50 μm)

Fig. 3

Media supplements added to hiPSC colonies to promote differentiation of mesodermal lineage. a In comparison to culture in basal conditions, RT-PCR demonstrated higher mRNA levels for brachyury (T) at day 2 (D2), along with similarly high mRNA levels for the mesoderm markers MIXL1 and CDX2 (ANOVA, *p < 0.05). Higher levels in mRNA for the node/notochord markers FOXA2, SHH, and Noggin observed at 3–5 days (D3–D5) after induction of differentiation as compared to culture in basal conditions (*p < 0.05). b Notochord marker NOTO was not upregulated at any time following induction of differentiation with FGF2 and BMP4; however, addition of Wnt-3a and Activin A promoted an early (D2) and sustained elevation (D3–D5) in mRNA for NOTO (*p < 0.05). This observation was key in our choosing to supplement colony cultures with Wnt-3a and Activin A at the earliest time points, days 1–3 (D1–D3). bFGF2 basic fibroblast growth factor, BMP bone morphogenetic protein 4, MIXL1 paired-type homeobox transcription factor identified in human, CDX2 member of the caudal-related homeobox transcription factor family, FOXA forkhead box protein A2, SHH sonic hedgehog, NOG noggin, NOTO notochord homeobox

Fig. 4

Images of hiPSC cultured for 36 days with nucleus pulposus differentiation media (NPDM) or basal media. a H&E staining of pellets cultured in NPDM (scale bar = 400 μM) or (b) basal medium showing formation of larger pellets with a vacuolated morphology when hiPSCs were promoted to differentiation in NPDM (scale bar = 400 μM). (c) When cultured in NPDM, human iPSCs contained vacuole-like structures (scale bar = 50 μm). d NPDM promoted expression of CD24, LM-α5, and BASP1 protein by 28 days of culture (scale bar = 50 μm). LMα5 alpha-5 subunit of heterotrimeric laminin, BASP1 brain abundant membrane attached signal protein 1

Fig. 5

Characterization of notochordal transcription factor brachyury expression via the T-fluorescent reporter construct. a Cells transfected with lentivirus containing plasmid for eGFP downstream of the brachyury (T) promoter. b Bright-field image and fluorescent image of a differentiated cell colony following culture in basal medium when supplemented with 40 ng/ml BMP4 and 20 ng/ml FGF2 (3 days) (scale bar = 100 μM). c eGFP expression detected via flow cytometry after supplementation with BMP4 and FGF2 at day 3 (showing 27.5% of cells for this population). d Before cell sorting, LV-T-GFP-positive cells were localized around the periphery of a differentiated cell colony (scale bar = 400 μM). e When cultured in basal medium supplemented with BMP4 and FGF2, flow cytometry analysis showed 25.4% of cells to be positive for T-GFP⁺ in differentiated hiPSCs. GFP green fluorescent protein, bFGF2 basic fibroblast growth factor, BMP bone morphogenetic protein 4

Fig. 6

NP markers enriched when differentiating human iPSCs sorted for T⁺ expression. a Immunohistochemistry showing higher expression of CD24, BASP1, and LMα5 of T-GFP⁺ sorted cells when cultured in NPDM as pellets (scale bar = 100 μM). b Safranin-O staining showing higher expression of GAGs in sorted T-GFP⁺ cells of pellets cultured in NPDM (scale bar = 50 μM). GFP green fluorescent protein, BASP1 brain abundant membrane attached signal protein 1, LMα5 alpha-5 subunit of heterotrimeric laminin