Generation of iPSC-derived limb progenitor-like cells for stimulating phalange regeneration in the adult mouse

Abstract

The capacity of digit tip regeneration observed both in rodents and humans establishes a foundation for promoting robust regeneration in mammals. However, stimulating regeneration at more proximal levels, such as the middle phalanges (P2) of the adult mouse, remains challenging. Having shown the effectiveness of transplantation of limb progenitor cells in stimulating limb regeneration in Xenopus, we are now applying the cell transplantation approach to the adult mouse. Here we report that both embryonic and induced pluripotent stem cell (iPSC)-derived limb progenitor-like cells can promote adult mouse P2 regeneration. We have established a simple and efficient protocol for deriving limb progenitor-like cells from mouse iPSCs. iPSCs are cultured as three-dimensional fibrin bodies, followed by treatment with combinations of Fgf8, CHIR99021, Purmorphamine and SB43542 during differentiation. These iPSC-derived limb progenitor-like cells resemble embryonic limb mesenchyme cells in their expression of limb-related genes. After transplantation, the limb progenitor-like cells can promote adult mouse P2 regeneration, as embryonic limb bud cells do. Our results provide a basis for further developing progenitor cell-based approaches for improving regeneration in the adult mouse limbs.

TAZ is required for chondrogenesis and skeletal development

“…Primary chondrocytes were isolated from the limb buds of embryo at E15.5. Briefly, the embryonic limb buds from WT, TAZ f/f , or Col2-Cre;TAZ f/f mice at E15.5 was dissociated with Trypsin solution (Fisher Scientific™, USA) by incubation for 30 min at 37 °C, and then washed the cells for two times with PBS and suspended in α-Minimum Essential Medium (α-MEM; Gibco, USA) 52 . Primary chondrocytes were cultured in α-MEM supplemented with Pen–Strep and 10% FBS (Gibco, USA).…”

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4

“…Thus we employed a 3D culture system amenable to mesoderm differentiation. Fibrin encapsulation supports maintenance of hPSCs 66 , cardiac mesoderm differentiation of hPSCs 67 , and LPM differentiation and subsequent limb-regenerating capability of mESCs 26 . Fibrin encapsulation here maintained the pluripotency of hiPSCs (Supplementary Table 2) and supported LPM differentiation with similar magnitude and time-course of marker expression as 2D culture (Fig.…”

“…LPM cells migrate to the limb fields and undergo condensation, proliferation, and ultimately chondrogenesis to cartilaginous elements that ossify to form the stylopod, zeugopod, and autopod skeletal elements of developing limbs 25 . Differentiation to a LPM-like cell phenotype was demonstrated with mouse 26 and human pluripotent stem cells 27,28 . Further, mESC aggregates that were differentiated to LPM-like cells engraft and contribute to developing limbs 29 and regenerating mouse phalanges 26 , highlighting the biological relevance of stem cell-derived LPM.…”

“…Differentiation to a LPM-like cell phenotype was demonstrated with mouse 26 and human pluripotent stem cells 27,28 . Further, mESC aggregates that were differentiated to LPM-like cells engraft and contribute to developing limbs 29 and regenerating mouse phalanges 26 , highlighting the biological relevance of stem cell-derived LPM. Using a protocol adapted from literature describing LPM differentiation via GSK3 inhibition 27 , LPM differentiation of hiPSCs over the course of 2 days here was characterized by induction of FOXF1 and loss of NANOG by qPCR and high content imaging.…”

Turning Nature’s own processes into design strategies for living bone implant biomanufacturing: a decade of Developmental Engineering

“…Those human progenitor cell populations displaying optimal characteristics for application in skeletal DE approaches could then be identified and isolated. Alternatively, it might be possible to derive these cell populations in vitro , as shown with murine iPSC-derived limb bud progenitors [ 141 ]. Noteworthy, preservation of the genomic, proteomic and regulatory landscapes (correct cellular identity) upon in vitro manipulation is key to proceed with DE approaches.…”