Background

Induced pluripotent stem cells (iPSC) have generated hope and excitement because of the potential they possess in regenerative medicine. Since the discovery by Yamanaka and colleagues that somatic cells can be reprogrammed to embryonic like state (ESC), numerous reports have emerged focusing on methods to generate them efficiently and safely for future clinical applications [1–7]. Some progress has been made toward development of techniques for generating safer iPSC; for example; generation of virus free iPSC, thus avoiding potential of viral effects on tumor formation, use of protein factors to reprogram somatic cells and reprogramming without using oncogenic factors [5–11]. In addition, efficiencies in reprogramming somatic cells have improved [12–15]. Although more work is still needed to get iPSC closer to clinical application, it is also critical to begin to understand factors that play a role in directing differentiation of iPSC to various cell lineages prior to clinical application. In this regard, several studies focusing on methods to direct iPSC to specific cell lineages are active areas of investigation [16–19]. These approaches emulate methods developed for directing ESC to various lineages [20–22]. Most studies have focused on directing ESC or iPSC to hematopoietic and or neural cells lineages [16–19]. As a proof of concept, iPSC were generated from a humanized mouse model of sickle cell anemia followed by correction of the sickle cell defect. iPSC with the corrected gene were then directed to hematopoietic cell lineage and given back to the somatic cell donor [18]. This proof of concept demonstrated that it was possible to direct iPSC to hematopoietic lineage efficiently at least for murine iPSC.

Directing human ESC or iPSC to neural lineages has also gained some success [17, 20, 23, 24]; Wernig and colleagues showed that iPS-cell-derived dopaminergic neurons could alleviate the disease phenotype in a rat model of Parkinson’s disease [17]. Differentiation of human embryonic stem cells (hESC) and or human induced pluripotent stem cells (hiPSC) to mesenchymal cell lineage have also been reported [53, 54]. After 28 days of incubation, cells were stained in Alizarin Red S solution and examined under a light microscope. Alizarin deposits were extracted with 10% acetic acid and used for quantification of mineralization.

Adipogenic differentiation

For adipogenic differentiation, iPSC-derived cells were plated in 12 well plates in adipogenic medium at a cell density of 5 × 103 cells per well. The adipogenic medium was composed of DMEM with high glucose supplemented with 10% FBS, 0.1 mM indomethacin, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 10-6 M dexamethasone. The media were replaced every 3 days for 28 days. Adipogenic differentiation was assessed by Oil Red O staining at 3 weeks after initial adipogenic induction. For Oil Red O staining, the cells were rinsed in PBS and fixed in 10% formalin followed by incubation of the cells in 2% (wt/vol) Oil Red O reagent for 5 minutes at room temperature, examined under light microscope and photographed. The cells were suspended in 0.5 ml of isopropanol to extract Oil Red O for quantification of the level of adipogenic differentiation.

In vivo bone formation

The Institutional Animal Care and Use Committee of Penn State University College of Medicine approved all animal procedures; all animal experiments were carried out following the approved protocol. Retinoic acid alone or in combination with TGF-beta1 iPSC derived cells were trypsinized and seeded onto HA/TCP ceramic scaffolds at 5 × 106 cells/mL. Cells were allowed to attach to the ceramics for 2 h at 37°C prior to implantation in animals. Cell seeded Scaffolds were implanted subcutaneously onto the backs of thymic SCID mice. Five weeks after implantation; animals were sacrificed and the scaffolds were harvested.

Histological analysis

For histological analysis, methods described previously were used [53]. Briefly, HA/TCP ceramic scaffolds seeded or not seeded with iPSC-derived cells and retrieved from recipient mice were fixed in freshly prepared 4% paraformaldehyde in PBS, containing 10% sucrose. Following fixation, the scaffolds were decalcified and embedded in paraffin. Ten micron tissue sections were cut, prepared for histological analysis and stained with H and E. Tissue sections were also stained by a modified Masson Trichrome Staining to demonstrate collagen synthesis [55].

Immunofluorescence for Osteocalcin and Dentin matrix protein

Cryosections prepared from the ceramic scaffolds seeded or not seeded with cells were retrieved from recipient mice at 5 weeks following implantation. Tissue sections were fixed in cold acetone for 5 minutes and treated with 10% goat serum, followed by treatment with a polyclonal antibody specific for Osteocalcin (1:20 Millipore) or DMP-1 (1:50 RDI). Tissue sections made from murine cortical bone were treated similarly. For visualization, sections were treated with the secondary rabbit anti-rat antibodies conjugated with either FITC (Millipore) or Rhodamine (Santa Cruz, Santa Cruz CA) at a concentration of 1:1000 and 1:500 respectively.

Gene expression analysis

Gene expression analysis of osteogenic and adipogenic associated genes were performed as described previously [53]. Triplicate PCR reactions were carried out.

Statistical analysis

Statistical analysis was carried out using SPSS® software (SPSS, Chicago, IL). One-way ANOVA with a Tukey’s post-hoc analysis was used to evaluate for differences in growth factors treated and untreated samples for osteogenic and adipogenic differentiation and marker expression. Significance was set at P < 0.05.