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Bulk RNA-seq of human nucleusus pulposus cell differentiations from embryonic stem cells and iPSCs

ABSTRACT: Progressive loss of nucleus pulposus cells (NPCs) is associated with the onset of intervertebral disc degeneration (IDD). Transplantation of NPCs, derived from human pluripotent stem cells including hESC/iPSCs, may offer a novel therapy for IDD. To date, effective in vitro differentiations of notochordal and NP cells remained to be demonstrated. Towards this end, we developed a three-step protocol to directly differentiate hESC/iPSC towards mesodermal, then notochordal and finally NPCs. Our results showed that notochordal-like cells (NCCs) were successfully derived from the first two-steps of the protocol. Furthermore, these cells could be differentiated into NPCs. These NPCs expressed the tyrosine kinase receptor Tie2 (Tie2), disialoganglioside 2 (GD2), collagen II and aggrecan. Genome-wide transcriptomic analyses by sequencing (RNA-seq) revealed the expression of a wide array of known NP markers, extracellular matrix (ECM) genes, up-stream regulators and pathways. Cross-comparison of in vitro RNA-seq profiles with in vivo human NP data confirmed the in vitro NPCs are significantly more similar to in vivo NP than hESC/iPSCs. Transplantation of NPCs effectively attenuated disc injury in a rat model of IDD. We utilized CRISPR/Cas9 to seamlessly knock in an enhanced green fluorescent protein (eGFP) to the loci of the Noto gene in ESCs for NCC generation. Our study achieved effective notochordal differentiation and provided transcriptomic insights into the use of human ESC/iPSCs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122429 | GEO | 2019/12/31

REPOSITORIES: GEO

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Project description:Intervertebral disc degeneration (IDD) leads to low back pain and disability globally. Progressive loss of nucleus pulposus cells (NPCs) are associated with the onset of IDD. Cell-based therapy has been shown the promising for many diseases, including the IDD in preclinical studies. However, the limited availability of human NPCs has hurdled such application for IDD. This study aimed to define strategies to derive NPCs from human ESC/iPSC. Human ESC3, ESC9 and IMR-90-iPSC were used for a three-step protocol to direct differentiation toward mesodermal, subsequently notochordal and finally NPCs. Our results showed that notochordal-like cells (NCCs) were successfully derived from the first two-step of protocol confirmed by immunofluorescence staining of Noto, Brachyury (T) and Foxa2. RT-PCR results showed that the expression of Noto, T and Foxa2 was the highest at day 5 after differentiation. Furthermore, these NCCs can be differentiated into NPCs via the final step of the protocol. These NPCs expressed the tyrosine kinase receptor Tie2 (Tie2), disialoganglioside 2 (GD2), collagen II and aggrecan. Genome-wide transcriptomic analyses by sequencing (RNA-seq) further reveals the expression of a wide array of extra-cellular matrix (ECM) genes, up-stream regulators and pathways. Cross-comparison of our RNA-seq profiles with public NPC data confirms the differentiated products are much closer to NPCs than are ESC/iPSC. Co-culturing NPCs with Light2 cell line, a luciferase-based reporter responsive to sonic hedgehog (Shh) secreted protein, showed NPCs synthesized Shh. Transplantation of NPCs effectively attenuated the spinal injury in a puncture-induced disc degeneration (PDD) model in rat. Furthermore, we utilized CRISPR/Cas9 technology to seamlessly knock in an enhanced fluorescent protein (GFP) to the loci of the noto gene in ESC9. Our study demonstrates that NPCs could be induced from human ESC/iPSC. This study provides a cell source for the development of novel strategy for IDD diseases.

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Bulk RNA-seq of human nucleusus pulposus cell differentiations from embryonic stem cells and iPSCs

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