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. 2017:2017:1513281.
doi: 10.1155/2017/1513281. Epub 2017 Jan 30.

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

Casey L Roberts  1 , Silvia S Chen  1 , Angela C Murchison  2 , Rebecca A Ogle  2 , Michael P Francis  1 , Roy C Ogle  3 , Patrick C Sachs  3
Affiliations

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

Casey L Roberts et al. Stem Cells Int. 2017.

Abstract

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells' origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2-6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPARγ but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells.

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Conflict of interest statement

The authors declare that there is no conflict of interests regarding the publication of this paper.

Figures

Figure 1
Figure 1
Characterization and reprogramming of donor-derived osteoblasts. (a) Example of donor bone chip in media (arrow) with osteoblasts emerging onto culture flask (asterisk). Osteoblasts were cultured in growth (b) or osteogenic induction media (c) and the resulting calcium deposits were stained with Alizarin Red S and imaged (scale bars = 30 μm and inserts are at 1x). (d) The donor-derived osteoblasts were assayed with qRT-PCR for osteoblast markers, osteocalcin, osteopontin, RUNX2, BMP2, and COL1A1 and compared to a commercially available osteoblast cell line (NHOst). (e) qRT-PCR analysis of pluripotent gene expression, Sox2 and Oct4, in the hOBs compared to neonatal BJ fibroblasts. (∗P < 0.05). (f) Alkaline phosphatase staining of primary reprogramming plate of hOB-iPSC. (g) TRA-1-81 immunofluorescence of initial hOB-iPSC colony (scale bars = 30 μm).
Figure 2
Figure 2
Pluripotent marker expression analysis of hOB-iPSCs and hFB-iPSCs. (a) hOB-iPSC and hFB-iPSC qRT-PCR expression of endogenous pluripotent genes, Sox2, Oct4, Nanog, and hTERT (∗P < 0.01). (b) Immunofluorescence of hOB-iPSC for pluripotency markers, TRA-1-60, Oct4, Nanog, Sox2, and IgG-FITC control (scale bars = 30 μm). (∗ indicates nonstaining MEF background.)
Figure 3
Figure 3
Differentiation of hOB-iPSCs into trilineages. (a) hOB-iPSC embryoid bodies (EBs) labeled with antibodies for cytoplasmic germ-layer markers, β-III tubulin (β-III; ectoderm), AFP (endoderm), and SMA (mesoderm). (b) hOB-iPSC EBs labeled with antibodies for germ-layer transcription factors, HAND1 (mesoderm), Sox1 (ectoderm), and Sox17 (endoderm). (c) Undetectable staining for pluripotent cells with TRA-1-60. Scale bars = 40 μm.
Figure 4
Figure 4
Induced osteoprogenitors (iOPs) cells differentiated from hOB-iPSC. (a) Flow cytometry analysis of iOPs and hMSCs revealed that the cells were positive for mesenchymal markers (CD29, CD44, CD90, CD105, and CD166) and negative for hematopoietic markers (CD14, CD31, and CD45) (black: CD marker expression, red: isotype control, and blue: unstained control). (b) Significantly (P < 0.05) faster growth kinetics of the hOB-iOPs and hFB-iOPs, as compared to hOBs and hMSCs in either 20% serum-containing media or Xenofree StemPro media at all time points. (c) Differentiation time of hOB-iPSCs to iOPs was assayed using flow cytometry for mesenchymal cell markers, CD44 and CD105. (d) iOPs were found to have similar levels of osteogenic related genes, RUNX2, BMP2, osteocalcin, and COL1A1 as compared to hMSCs ∗ indicates significant difference from the hOB-iOPs (P < 0.05). iOPs downregulated all pluripotency genes, Oct4, Sox2, Nanog, and hTERT indicating successful differentiations. ∗ indicates significant difference from the iPSC (P < 0.05).
Figure 5
Figure 5
iOP differentiation to adipocytes, chondrocytes, and osteoblasts. (a) Gene expression of adipocyte, chondrocyte, and osteoblast markers normalized to their respective growth media control cells expression levels. Adipogenic expression levels were lower than both hFB-iOP and hMSC indicating a lack of adipogenic capacity  ∗Significantly different from hOB-iOP differentiated cells (P < 0.05). (b) Alizarin Red S, Oil Red O, and Alcian blue staining of adipogenic, osteogenic, and chondrogenic differentiations with a hematoxylin counterstain demonstrated comparable chondrogenic and osteogenic differentiation capacity, with a lack of hOB-iOP adipogenesis. Scale bars = 30 μm.
Figure 6
Figure 6
Promoter methylation analysis for unreprogrammed, parental iPSC, and iOP cells. Methylation sensitive enzymatic digestions were performed for hFB and hOB (a), hFB-iPSC and hOB-iPSC (b), and hFB-iOP and hOB-iOP (c). Graphs indicate promoter percent methylated (black) versus percent unmethylated (gray) of a subset of genes: endoderm (FOXA2, STAT1), ectoderm (OLIG2), and mesoderm (NOTCH2, GATA2, PPARγ, RUNX1, and RUNX2).
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References

    1. LoGuidice A., Houlihan A., Deans R. Multipotent adult progenitor cells on an allograft scaffold facilitate the bone repair process. Journal of Tissue Engineering. 2016;7 doi: 10.1177/2041731416656148. - DOI - PMC - PubMed
    1. Musante D. B., Firtha M. E., Atkinson B. L., Hahn R., Ryaby J. T., Linovitz R. J. Clinical evaluation of an allogeneic bone matrix containing viable osteogenic cells in patients undergoing one- and two-level posterolateral lumbar arthrodesis with decompressive laminectomy. Journal of Orthopaedic Surgery and Research. 2016;11(1, article 63) doi: 10.1186/s13018-016-0392-z. - DOI - PMC - PubMed
    1. Vanichkachorn J., Peppers T., Bullard D., Stanley S. K., Linovitz R. J., Ryaby J. T. A prospective clinical and radiographic 12-month outcome study of patients undergoing single-level anterior cervical discectomy and fusion for symptomatic cervical degenerative disc disease utilizing a novel viable allogeneic, cancellous, bone matrix (trinity evolution™) with a comparison to historical controls. European Spine Journal. 2016;25(7):2233–2238. doi: 10.1007/s00586-016-4414-7. - DOI - PubMed
    1. Lennon D. P., Schluchter M. D., Caplan A. I. The effect of extended first passage culture on the proliferation and differentiation of human marrow derived mesenchymal stem cells. Stem Cells Translational Medicine. 2012;1(4):279–288. doi: 10.5966/sctm.2011-0011. - DOI - PMC - PubMed
    1. Mareschi K., Ferrero I., Rustichelli D., et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow. Journal of Cellular Biochemistry. 2006;97(4):744–754. doi: 10.1002/jcb.20681. - DOI - PubMed

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