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Osteoblasts Derived from Induced Pluripotent Stem Cells form Calcified Structures in Scaffolds both in vitro and in vitro

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From the February 2011 Issue of Stem Cells

Paper commentary by Stuart Atkinson

The correct and efficient differentiation of pluripotent cell types to clinically relevant cell types is a major common goal in stem cell biology. Recent advances in induced pluripotent stem cell (iPSC) technologies have put the prize of patient-specific stem cells for autologous cellular therapy firmly within biomedical sciences’ grasp. The regeneration of the musculoskeletal system is one such system in which iPSC technology could have a great impact. Current strategies involve bone autografts and autologous transplantation of mesenchymal stem cells (MSCs) from the bone marrow, but both have severe limitations. Bone autografts are invasive and have a high morbidity rate (Arrington et al.) and while autologous MSCs exhibit great potential for musculoskeletal regeneration, their proliferative potential decreases greatly with age, perhaps limiting this type of therapy to younger patients (Stenderup et al.). The potential of iPSCs to be differentiated to MSCs has been demonstrated previously (Lian et al.), and indeed these iPSC-derived MSCs were shown to contribute to tissue regeneration more than bone marrow-derived MSCs in a rodent model of hind limb ischemia. Moreover, the MSCs generated in this particular study were able to proliferate for 120 population doublings with no observable karyotypic abnormalities, overall making iPSC-derived MSCs a very attractive proposition. Now, Bilousova et al. from the laboratory of Susan M. Majka at the University of Colorado, Denver in the forthcoming edition of Stem Cells extend these previous studies to further evaluate the potential of iPSCs for use in the regeneration of the musculoskeletal system.

iPSCs were produced from mouse fibroblasts using retroviruses encoding for Oct3/4, Sox2, Klf4 and c-Myc, then were induced to differentiate towards mesenchymal lineages. Importantly, Bilousova et al. performed an additional step compared to Lian et al. by initially culturing their iPSCs as embryoid bodies (EBs) for a short period of time in the presence of all-trans retinoic acid (ATRA) in order to induce mesenchymal differentiation of the iPSCs to enhance the capability of these cells to later form mature mesenchymal cell types using various protocols. The subsequent research went on to show that these mesenchymally induced iPSCs could indeed readily differentiate into mesenchymal cell types. Fat differentiation (scored by visualisation of lipid droplet accumulation) of the mesodermally induced iPSCs was induced at 4 weeks of iPSC differentiation on gelatin coated plates while cartilage differentiation was established by culturing EBs of the mesodermally induced iPSCs as non-adherent cell spheres in chondrogenic medium. Differentiation towards a chondrogenic fate was demonstrated by the culture of the mesodermally induced iPSCs in chondrogenic medium for 5 weeks, as a monolayer on gelatin. This led to the expression of the chondrogenic differentiation factor Sox9 and the chondrocyte matrix protein Acan (Aggrecan). Alcain blue staining, which highlights extracellular matrix rich in polyanionic glycosaminoglycans, and multiple mesenchymal markers (CD90, 73, 105, 106 and 133) were present which, combined with the lack of haematopoietic markers (CD45 and c-Kit), suggest a chondrocytic phenotype.

Differentiation into osteoblasts was achieved by culturing mesodermally induced iPSC in osteogenic medium for up to 8 weeks. An osteoblastic phenotype was confirmed using specific histochemical stains (alizarin red staining demonstrates calcium deposition and mineralisation while von Kossa stains phosphate deposition) and specific mRNA markers (Runx2, Col1a1, Spp1 and Flt1). The acid test to evaluate the use of these cells in bone formation is the ability to maintain their differentiated osteoblast phenotype when seeded into 3D scaffolds, such as gel-foam carriers which are biodegradable gelatin matrixes. In vitro analysis at 48 hours and 2 weeks post implantation of the cells into the gel-foam carrier, showed an increase in osteoblast cell number and matrix secretion at 2 weeks, maintenance of Runx2, CollA1 and Spp1 expression at 48 hours and increased Colla1 and Spp1 at 2 weeks. In vivo analysis utilised gel-foam cubes seeded with cells that were subcutaneously implanted on the dorsal aspect of immunocompetent ICR (Imprinting Control Region) mice. After 12 weeks, analysis showed no tumour formation, evidence of recruitment of host derived vasculature (rather than vasculature derived from the implanted cells) and staining demonstrated the presence of calcium in the implants and the presence of osteoblast markers, all suggestive of some in vivo functionality of these cells.

Overall, Bilousova et al.are the first to show that iPSC-derived osteoblasts can maintain their phenotype when cultured on a 3D scaffold both in vitro and in vivo. These results therefore show much promise for iPSC-mediated regeneration of the musculoskeletal system. These are also encouraging results in light of another studies showing that iPSC-derived cells (hemangioblasts and retinal pigmented epithelium cells) have limited expansion capability and senesce very early post-differentiation, therefore suggesting that iPSC-derived cells for cellular therapy would be of little use (Feng et al.).

 

References

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Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA.
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Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells.
Stenderup K, Justesen J, Clausen C, Kassem M.
Bone. 2003 Dec;33(6):919-26.

Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice.
Lian Q, Zhang Y, Zhang J, Zhang HK, Wu X, Zhang Y, Lam FF, Kang S, Xia JC, Lai WH, Au KW, Chow YY, Siu CW, Lee CN, Tse HF.
Circulation. 2010 Mar 9; 121(9):1113-23.

Bilousova G, Hyun Jun D, King KB, Delanghe S, Chick WS, Torchia EC, Chow KS, Klemm DJ, Roop DR, Majka SM.
Osteoblasts Derived from Induced Pluripotent Stem Cells form Calcified Structures in Scaffolds both in vitro and in vitro.
Stem Cells. 2010

Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence.
Feng Q, Lu SJ, Klimanskaya I, Gomes I, Kim D, Chung Y, Honig GR, Kim KS, Lanza R.
Stem Cells. 2010 Apr;28(4):704-12.

 

Further Reading
Induced Pluripotent Stem (Ips) Cells and Lineage Reprogramming: Prospects for Bone Regeneration.
Illich DJ, Demir N, Stojković M, Scheer M, Rothamel D, Neugebauer J, Hescheler J, Zöller JE.
Stem Cells. 2011