Background/Purpose: Many genetic factors and cell types contribute to the axial inflammation, trabecular bone loss, and aberrant bone formation that result in ankylosing spondylitis. The functional consequences of genetic variants may be manifest in specific cell types and in many cases affected cell types are not readily accessible for evaluation. In order to obtain these cell types we examined the feasibility of reprogramming fibroblasts from patients with axial spondyloarthritis (AxSpA) into induced pluripotent stem cells (iPSCs) and subsequent re-differentiating  iPSCs into various lineages.

Materials and Methods: Dermal fibroblasts from 7 AxSpA patients and 3 healthy controls (HC) were reprogrammed using a Sendai virus vector encoding Oct4, Sox2, Klf4 and Myc. One AxSpA and one HC fibroblast line were reprogrammed in two different labs to assess technical reproducibility. Virus-free iPSCs were differentiated into mesenchymal stem cells (MSCs) using a TGF-β inhibitor. MSCs were differentiated into osteoblasts, chondrocytes, and adipocytes; defined cytokine cocktails were used to differentiate iPSCs into monocytes. IPSC derived monocytes were treated with RANKL to induce osteoclastogenesis. IPSC gene expression patterns were established by RNAseq. Gene expression during MSC-osteoblast differentiation was monitored by Nanostring. Flow cytometry was conducted to evaluate the expression of iPSC, MSC and monocyte specific markers.

Results: All iPSC lines expressed  pluripotency markers (OCT4, TRA-1, SSEA-4) and stem cell specific genes. IPSC derived MSCs proved positive for CD105, CD73, CD90 and CD44, but lacked CD45, CD34, CD11b, CD19, and HLA-DR. MSCs exhibited the capacity to induce differentiation of peripheral blood monocytes into osteoclasts in co-culture as demonstrated by tartrate resistant acid phosphatase staining. MSCs could be differentiated into mineralizing osteoblasts, as confirmed by expression of osteogenic genes and Alizarin-Red staining, into chondrocytes (proteoglycan staining with Alcian-Blue), and into adipocytes (lipid staining with Oil Red O). iPSC-derived monocytes/macrophages expressed HLA-DR, CD14, CD86, CD80 and CX3CR1 and CD45, and were capable of phagocytosing beads and differentiating into osteoclasts. Preliminary comparison of mineralization potential revealed that MSCs from AxSpA patients (n=3) exhibited 3-fold higher mineralization than the HC as determined by Alizarin Red staining. Independently-derived iPSC lines behaved similarly.

Conclusion: We have successfully derived iPSCs from AxSpA patient fibroblasts. The iPSCs can be differentiated into functional MSCs capable of differentiating into mature osteoblasts, chondrocytes, and adipocytes, as well as inducing monocytes to become osteoclasts. We have also successfully generated hematopoietic cells that can differentiate into monocytes, macrophages and osteoclasts. Notably, MSCs from AxSpA patients demonstrated greater mineralization capacity. This was observed repeatedly in independently iPSC derived MSCs from the same patient. These cells may provide a powerful system to examine the molecular functional consequences of genetic differences that predispose to SpA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/induced-pluripotent-stem-cells-ipscs-as-a-tool-for-unraveling-the-role-of-different-cell-types-in-the-disease-process-of-spondyloarthritis-pathogenesis/