Engineering phenotypically stable bone-like tissue from human embryonic stem cells 1,2Marolt, D; 1Marcos Campos, I; 1Bhumiratana, S; 1Koren, A; 1Petridis, P; 3Grayson, W L; 1Spitalnik, P; +1Vunjak Novakovic, G

+1Columbia University, New York, NY, 2The New York Stem Cell Foundation, New York, NY, 3Johns Hopkins University, Baltimore, MD gv2131@columbia.edu

INTRODUCTION: Repair of large bone defects remains limited by the ability to harvest and shape autologous bone grafts, or by the non-biological nature of bone substitutes. Bone tissue engineering could provide an unlimited supply of functional viable bone grafts. Human embryonic stem (ESC) cells, and the embryonic-like iPS cells, represent a promising cell source for this goal, as they can: (i) grow indefinitely, providing unlimited numbers of tissue repair cells, and (ii) give rise to any cell type in the body. Osteogenic cells have previously been derived from ESC1, however their ability to form three-dimensional bone tissue in vitro or in vivo has not been demonstrated2,3. In the current study, we have tested whether an in vitro model of bone development utilizing decellularized bovine bone scaffolds and perfusion bioreactors could be applied to ESC. We hypothesized that hESC-derived mesenchymal-like progenitors will form bone-like tissue under tissue engineering conditions optimized for human mesenchymal cells from bone marrow (BMSC)4. To test this hypothesis, we investigated bone formation by ESC and BMSC over 5 weeks of culture, and its phenotypic stability over 8 weeks in vivo. METHODS: ESC differentiation and characterization: Human ESC lines H9 and H13 (Wicell Research Institute) were induced to differentiate in KnockOut DMEM with 10% FBS for 7 days, trypsinized and seeded to gelatin-coated tissue culture plates (“passage 1”). Adherent cells were subcultured, and used at passage 4 for characterization assays and construct cultivation. BMSC (Lonza) served as controls. Cultivation of bone constructs: ESC-progenitors and BMSC were seeded on fully decellularized bovine trabecular bone scaffolds (4 mm Ø x 4 mm), and cultured in osteogenic medium (DMEM with 10%FBS, beta-glycerophosphate, dexamethasone and ascorbate-2 phosphate), statically or in perfusion bioreactors providing interstitial flow through the developing tissues, for 5 weeks. Interstitial velocity of 800 µm/sec was selected based on our previous studies with BMSC4. In vivo study: Bioreactor-cultured constructs and cell-seeded scaffold controls were implanted subcutaneously in scid-beige mice for 8 weeks. Assessments: Cell viability was determined by a live/dead assay after seeding and after 3 and 5 weeks of culture. DNA content, alkaline phosphatase activity and osteopontin release into culture medium were measured to assess cell growth and osteogenesis. Bone tissue formation was assessed by H&E, Masson Trichrome, osteopontin, bone sialoprotein and osteocalcin stainings, and by µCT imaging.

Figure 1: Osteogenesis of ESC (H9) and BMSC. Cell numbers (A), alkaline phosphatase (AP) activity (B) and osteopontin release (C) are shown (P<0.05;*st vs br; #ESC vs. BMSC; $wk3 vs. wk5). Tissue formation positive for osteocalcin stain is shown for H9 (D-E, G-H) and BMSC (F, I) cultured statically (st) (D, G) and in bioreactors (br) (E-F, H-I) at 3 (D-F) and 5 (G-I) weeks. Inset: negative control. RESULTS: Cell characterization: Both ESC lines were differentiated into progenitors that proliferated steadily over 10 passages, expressed mesenchymal surface antigens (>85% positive for CD44, CD73, CD90, CD166) and exhibited strong osteogenic (AP activity, matrix mineralization), and weak chondrogenic (GAG deposition) and adipogenic (lipid vacuoles deposition) potentials (data not shown).

Bone tissue engineering and in vivo stability: Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into culture medium as compared to static cultures (Fig.1A-1C). Histological examination revealed formation of dense extracellular matrix in the bioreactors, as evidenced by the presence of bone matrix proteins osteopontin, bone sialoprotein and osteocalcin (Fig.1D-1I). In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce (Fig.1D, 1G). µCT revealed mineralized tissue formation during the 5-week culture in all groups (Fig.2A-B). Osteogenesis and bone tissue formation were comparable for ESC and BMSC.

Figure 2: Bone tissue formation in vitro and stability in vivo. (A) µCT scans of H9-constructs cultured in bioreactors. (B) Increasing mineralized tissue volume (BV) and bone to total construct volume (TV) fraction (P<0.05;*group vs. initial; $group vs. wk5). (C) H&E stain showing bone and control scaffolds (with H9 teratoma) after explant.

After 8-weeks of subcutaneous implantation, further maturation of engineered bone was noted (Fig.2A-B), resulting in denser bone matrix compared to scaffolds seeded with progenitors prior to implantation (Fig.2C). Importantly, there was no evidence of teratoma tissue formation, which was found as expected in ESC-seeded scaffolds (Fig.2C). Engineered bone constructs contained microvasculature spanning interior regions of the scaffolds, and initiation of remodeling (evidenced by the presence of osteoclastic cells in the outer regions). DISCUSSION: Our study shows that compact bone grafts can be engineered from ESC-derived mesenchymal progenitors using the same scaffolds and bioreactor cultivation conditions as with BMSC. Engineered bone properties were similar for the two cell sources. Importantly, stepwise ESC differentiation and bone engineering protocol yielded stable bone tissue with potential for further maturation and integration in vivo. SIGNIFICANCE: Our results provide basis for development of clinical-size bone grafts from ESC, as well as models for advanced quantitative studies of bone development by recapitulating some aspects of native tissue in vitro. ACKNOWLEDGEMENTS: This work was supported by NYSCF (DM is a NYSCF-Helmsley Investigator) and NIH (grants DE016525 and EB002520). REFERENCES: 1. de Peppo GM, et al. Tissue Eng Part A, 2010, 16(11):3413 2. Kim S, et al. Biomaterials, 2008, 29(8):1043 3. Kuznetsov SA, et al. Stem Cells Dev, 2011, 20(2):269 4. Grayson WL, et al. Biotechnol Bioeng, 2011, 108(5):1159

Paper No. 0002 • ORS 2012 Annual Meeting