Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells.

Tomoka Takao,Toki Kitano,Eiji Nakata,Takeshi Takarada,Masahiro Nakamura,Daisuke Yamada

doi:10.3390/ijms23052661

Abstract

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1+ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1+ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato+ LBM-like cells are defined as CD44high CD140Bhigh CD49f−. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44high CD140Bhigh CD49f− PRRX1+ LBM-like cells.

Highlights

As the appendicular skeleton or limbs develop, lateral plate mesoderm (LPM) cells differentiate into paired related homeobox 1 (PRRX1)+ limb-bud mesenchymal (LBM) cells [12], and LBM-derived osteochondroprogenitors form articular cartilage and bone tissue [13]
Using a PRRX1-tdTomato reporter Human pluripotent stem cells (hPSCs) line, we showed that CD44high CD140Bhigh CD49f− can be used to identify PRRX1-tdTomato+ LBM-like cells
We cultured PRRX1-tdTomato reporter hPSCs for two different time periods—three days and seven days— during the pluripotent state to determine their effect on inducing PRRX1+ LBM-like cells

Summary

Introduction

Damaged cartilage is a hallmark of osteoarthritis and rheumatoid arthritis damage cartilage. Cartilage transplantation therapy aims to regenerate damaged cartilage [1,2], current methods, including microfracture and osteochondral autograft transplantation, are invasive and burdensome for patients. Human cartilage tissues develop from neural crest (NC), paraxial mesoderm (PM)derived, or lateral plate mesoderm (LPM)-derived lineages, which each exists in the cranial, axial, and appendicular skeleton, respectively [3,4]. Human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), are a versatile cell resource for basic research and tissue regeneration because of their indefinite proliferation and pluripotency [5]. Several groups have induced iMSC-, NC-, or PM-derived hyaline cartilage tissues from hPSCs [6–11]. As the appendicular skeleton or limbs develop, LPM cells differentiate into paired related homeobox 1 (PRRX1)+ limb-bud mesenchymal (LBM) cells [12], and LBM-derived osteochondroprogenitors form articular cartilage and bone tissue [13].

Methods

Results

Conclusion