Induced pluripotent stem cell technology in bone biology

Abstract

Technologies on the development and differentiation of human induced pluripotent stem cells (hiPSCs) are rapidly improving, and have been applied to create cell types relevant to the bone field. Differentiation protocols to form bona fide bone-forming cells from iPSCs are available, and can be used to probe details of differentiation and function in depth. When applied to iPSCs bearing disease-causing mutations, the pathogenetic mechanisms of diseases of the skeleton can be elucidated, along with the development of novel therapeutics. These cells can also be used for development of cell therapies for cell and tissue replacement.

Keywords: Induced pluripotent stem cells R; Models of disease; Osteogenic differentiation; Pluripotent stem cells; Reprogramming.

Fig 1.

Stem cell self-renewal – a defining characteristic of a stem cell. A. There are several ways that a stem cell may divide. In one case, a stem cell divides asymmetrically, with one daughter remaining a stem cell, and the other becoming more specified, and may transiently amplify before becoming terminally differentiated. Or a stem cell may divide symmetrically, and in one case, both daughters are committed, which would lead to stem cell loss, and in the other case, both daugters are both stem cells, which may lead to immortalization. The actual process of stem cell maintenance is not yet well understood, and may rely on toggling between the different types of division. B. An assay to demonstrate self-renewal of a skeletal stem cell present in bone marrow. A single cell from bone marrow with a defined phenotype (CD45⁻/CD34⁻/CD146⁺) is isolated by FACS and allowed to form a colony. When the colony is transplanted in vivo, the progeny of that single cell gives rise to a bone/marrow organ (ossicle). A single cell with the same original phenotype (CD146⁺) is isolated from that ossicle, and is able to give rise to a secondary colony, that when transplanted is able to form a secondary ossicle with the same CD146⁺ phenotype (a pericyte) (modified from Sacchetti et al, Cell, 2007).

Fig 2.

Development and stem cell hierarchy and utilization. A. Development starting from the fertilized egg (totipotent), going through the blastocyst stage (pluripotent), and then gastrulation, which establishes the three germ layers (multipotent). B. Pluripotent stem cells can be transplanted into mouse embryos (MOUSE ONLY), and will give rise to all cells of the zygote, embryo and fetus. This was a major advance in mouse genetics. Pluripotent stem cells can also differentiation into all cell types in vitro, and can form embryoid bodies, which can exhibit cells from all three germ layers. However, the gold standard to demonstrate pluripotency is by in vivo transplantation, which leads to formation of a teratoma that contains derivatives of all three germ layers.

Fig 3.

Development of bone. A. Following gastrulation and formation of the three different germ layers, the ectoderm gives rise to neural crest cells that migrate away from the neural tube, some of which form the frontal bones of the skull, and the facial bones. The sclerotome (the ventral part of somites made of paraxial mesoderm) forms the axial skeleton, and the lateral plate somatic mesoderm forms the appendicular skeleton. B. There are diseases that specifically affect bones from germ layer specific elements.

Fig 4.

Methods of reprogramming somatic cells. A. Somatic nuclear cell and chromosomal transfer. B. Use of viral vectors and other modes of introducing four transcription factors (OSKM) into somatic cells of varying types. Figure modified from Bischoff, SR, NovoHelix https://www.novohelix.com/cell%20line%20models/pluripotent-stem-cells-reprogramming).

Fig 5.

Differentiation of hiPSCs into lineage-specific osteoprogenitor cells from three different embryonic origns (paraxial mesoderm, lateral plate mesoderm and neural crest (Kidwai et al, Stem Cells, 2020).

Fig 6.

In vivo transplantation of lineage-specific osteoprogenitor cells into immunocompromised mice (NSG) in combination with a ceramic scaffold (Attrax^™) after 16 weeks of transplantation. Bone formation was verified to be of donor origin based on identification of human cells by human ALU in situ hybridization (Kidwai et al, Stem Cells, 2020).