Generation and differentiation of induced pluripotent stem cells reveal ankylosing spondylitis risk gene expression in bone progenitors

Gerlinde Layh-Schmitt,Stephen R Brooks,Cristina Montagna,Massimo Gadina,Kathryn M Davis,Robert A Colbert,Emily Lazowick,Fatemeh Navid,Shajia Lu

doi:10.1007/s10067-016-3469-5

Abstract

Axial spondyloarthritis (axSpA), which encompasses ankylosing spondylitis, is a complex genetic disease. Aberrant bone formation is a key feature of pathogenesis that can lead to ankylosis of the spine. Our objective is to determine, whether genes whose variants confer susceptibility to AS are expressed in bone progenitors like mesenchymal stem cells (MSCs). Since MSCs from bone marrow is difficult to obtain, we first examined, whether MSCs can be derived from induced pluripotent stem cells (iPSCs). Dermal fibroblasts of two axSpA patients and one healthy control were reprogrammed into iPSCs using a Sendai virus vector encoding pluripotency genes. Pluripotency of iPSCs was examined by embryoid body formation and by testing for stem cell specific gene and protein expression using RT-PCR and immuno fluorescence. iPSCs were differentiated into MSCs by a TGFß inhibitor. MSCs were characterized by flow cytometry using lineage specific antibodies and by their capacity to develop into chondrocytes, adipocytes, and osteoblasts in lineage-specific medium. RNA-seq was applied to determine genome-wide gene expression patterns in MSCs, iPSCs, and blood. We show for the first time, that expression levels of several AS susceptibility genes (EDIL3, ANO6, HAPLN1, ANTXR2) involved in bone formation are significantly elevated in MSCs (2–15-fold; p ≤ 0.05) compared to blood or iPSCs and demonstrate that iPSC-derived MSCs can be differentiated into osteoblasts, chondrocytes, and adipocytes. We conclude, MSCs generated from patient fibroblast-derived iPSC lines are useful tools for studying functional genomics of risk genes associated with bone formation in AS pathogenesis.

Highlights

Ankylosing spondylitis (AS) is characterized by inflammation of the sacroiliac joints, vertebral bodies and facet joints, entheses, and peripheral joints, as well as extra-articular sites such as the gastrointestinal tract [1]
Embryoid body (EB) generated from induced pluripotent stem cells (iPSCs) exhibited all three germ layers based on expression of the endodermal marker AFP, the mesodermal protein SMA, and the ectodermal marker βIII-TUB (Fig. 1b, right panel) [12, 16]
Progress in understanding the pathogenesis of structural damage in Axial spondyloarthritis (axSpA)/AS has been hampered by several factors, including the anatomic location of affected tissue, the absence of animal models that faithfully reproduce the bone phenotype, and the lack of primary patient-derived cell lineages that mediate osteoproliferation and bone marrow edema

Summary

Introduction

Ankylosing spondylitis (AS) is characterized by inflammation of the sacroiliac joints, vertebral bodies and facet joints, entheses, and peripheral joints, as well as extra-articular sites such as the gastrointestinal tract [1]. Mechanisms underlying the pathogenesis of axSpA/AS, and in particular the involvement of specific cell types and events that lead to bone marrow edema, loss of trabecular bone, and aberrant bone formation in the spine, are still poorly understood [4, 5] Evidence that cytokines such as TNFα [6], IL-23 [7], and IL-17 [8] are expressed in axial skeletal tissue from AS patients has provided a rationale for current therapies [9,10,11]; but the mechanisms driving their production and their effects on bone formation require further study. Progress in this area has been hampered by limited access to cell types that contribute directly to disease pathogenesis, such as hematopoietic progenitors, mesenchymal cells, and their derivative osteoblasts and adipocytes

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