Regeneration of the human segmentation clock in somitoids in vitro

Abstract

Each vertebrate species appears to have a unique timing mechanism for forming somites along the vertebral column, and the process in human remains poorly understood at the molecular level due to technical and ethical limitations. Here, we report the reconstitution of human segmentation clock by direct reprogramming. We first reprogrammed human urine epithelial cells to a presomitic mesoderm (PSM) state capable of long-term self-renewal and formation of somitoids with an anterior-to-posterior axis. By inserting the RNA reporter Pepper into HES7 and MESP2 loci of these iPSM cells, we show that both transcripts oscillate in the resulting somitoids at ~5 h/cycle. GFP-tagged endogenous HES7 protein moves along the anterior-to-posterior axis during somitoid formation. The geo-sequencing analysis further confirmed anterior-to-posterior polarity and revealed the localized expression of WNT, BMP, FGF, and RA signaling molecules and HOXA-D family members. Our study demonstrates the direct reconstitution of human segmentation clock from somatic cells, which may allow future dissection of the mechanism and components of such a clock and aid regenerative medicine.

Canalization and competition: the cornerstone of genetic network’s dynamic stability and evolution

“…This mechanism assumes a significant role in biological processes. For example, during human segmentation development, antagonistic gene pairs associated with WNT, FGF, and Notch pathways collectively contribute to the formation of paraxial mesoderm segmentation and the establishment of segmental clock [25, 26]. Signals within this pathway are crucial for maintaining an overall regulatory trend, which holds significant biological implications in terms of monotonic modulation.…”

“…The formation of body segments along with the anterior-posterior axis is facilitated by concentration gradients of growth factors. The induced selfsustaining oscillations also necessitates the coordination between global signaling and local states [24][25][26]. The hidden mechanism, known as monotonicity, refers to the well-organized set of positive and negative consistent regulations that form sustaining or oscillating signal patterns [27].…”

Generation of musculoskeletal cells from human urine epithelium-derived presomitic mesoderm cells

“…Consistent with the in vivo development of presomitic mesoderm [ 9 , 11 , 52 ], UiPSM cells are capable of differentiating into skeletal myocytes, osteoblasts, and chondroblasts in vitro. Our prior work has shown that UiPSM cells, reprogrammed from urinary cells (UCs) and exiting the pluripotent state, retain somatic stemness, thereby mitigating the risk of tumorigenesis [ 10 ]. This suggested the potential of UiPSM cells to serve as a developmental model for musculoskeletal research.…”

“…Refer to the paper [ 10 ]. Human urine derived epithelioid cells were electrical transduced unintegrated plasmids (pEP4EO2SET2K and pCEP4-miR-302–367 cluster) using Amaxa™ Basic Nucleofector™ Kit (Lonza, VPI-1005), then cultured 7–12 days in the induction medium (IM: advanced DMEM/F12 (Gibco, 12,634–010) supplemented with 3 µM CHIR99021 (Synthesized in GIBH), 10 ng/ml bFGF (PeproTech P09038), 5 ng/ml EGF (R&D systems, 236-EG) and 5 µM EPZ5676 (Selleck Chemicals S7062)), changed the medium every 2 days.…”

“…In our research, we have successfully reprogrammed human urinary epithelial cells into presomitic mesoderm progenitor cells (UiPSMs). These cells are capable of long-term expansion in vitro and differentiate into mesoderm cell types in vivo, displaying anteroposterior axis and segmentation clock features [ 10 ]. In this study, we established a UiPSM-based approach to skeletal myogenesis, enhanced by MYOD-mediated maturation.…”