Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED) and MSC-like cells from iPSCs from SHED (iPS-SHED) and from human dermal fibroblasts (iPS-FIB). MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios.


Introduction
Clinical demand for bone tissue is evident to supplant bony structures lost due to trauma, disease, or congenital malformation. Cell replacement therapies represent a promising strategy for bone engineering, and human mesenchymal stem cells (MSCs) isolated from various adult tissues have been extensively investigated as a potential cell source for bone regenerative treatments [1,2]. However, large-scale applications are constrained since MSCs are found in limited amounts, are highly heterogeneous, and their long-term in vitro expansion can lead to senescence and spontaneous differentiation [3,4]. Additionally, the differentiation potential of MSCs may vary depending on the tissue of origin [5].
Generation of human induced pluripotent stem cells (hiPSCs) was first achieved using dermal fibroblasts [6,7]. Thereafter, hiPSCs have been derived from an ample variety of starting cells, including MSCs. Reprogramming MSCs to hiPSC is an attractive approach to circumvent issues associated with the direct use of MSCs since it allows the production of cells with robust in vitro self-renewal capacity and with differentiation multipotential. Controlling differentiation cues in vivo is a significant challenge and direct transplantation of pluripotent stem cells may result in tumor formation [8]. Therefore, derivation of MSC-like cells from 2 Stem Cells International pluripotent stem cells has been pursued by a number of researchers [9][10][11].
Most types of MSCs are not easily obtained using minimally invasive procedures. Stem cells from human exfoliated deciduous teeth (SHED) can be easily isolated from a readily accessible tissue source, expanded under simple culture conditions, and banked. Even though SHED have been reported to be especially useful to restore bone [12,13], as mentioned above, their inherent population heterogeneity and limited expansion capacity restrict their use for therapeutic purposes. While hiPSCs have been generated from SHED (iPS-SHED) [14], there is no report exploring the in vitro osteogenic potential of MSC-like cells derived from iPS-SHED populations. Therefore, the goal of this study is threefold: (1) to verify if MSC-like cells from iPS-SHED and SHED isolated from the same donors exhibit similar in vitro osteogenic potential; (2) to compare the osteogenic potential of MSC-like cells from iPS-SHED with MSC-like cells from hiPSCs derived from mature dermal fibroblasts (iPS-FIB), considered the most accessible cell source for iPSC generation; (3) to compare the expression of CD105 between these cellular populations, which has been inversely correlated with an increased osteogenic potential [15].

Isolation of Stem Cells from Human Exfoliated Dental Tissue (SHED), Human Dermal Fibroblasts, and Generation of Human Induced Pluripotent Stem Cells (hiPSCs)
. SHED were obtained from teeth of 6 independent subjects by enzymatic digestion of pulp from deciduous teeth as described in Miura et al., 2003 [12]. Human adult dermal fibroblasts, the most accessible and feasible cell source for iPSC generation [14], were obtained according to the protocol detailed in Aasen and Belmonte 2010, adapted for fibroblast isolation [16]. hiPSCs were obtained from SHED from 2 independent subjects (3 clones derived from each) and fibroblast cell populations from 3 independent subjects (2 clones each). SOX2, c-MYC, OCT4, and KLF4 ectopic expression were induced through retroviral transduction, as originally reported in Takahashi et al., 2007 [6]. Two days after transduction, SHED and fibroblasts were cocultivated with irradiated murine embryonic fibroblasts (Millipore) in embryonic stem cell medium Dulbecco's modified Eagle/F12 medium (DMEM/F12) supplemented with 2 mM GlutaMAX-I, 0.1 mM nonessential amino acids, 55 uM 2-mercaptoethanol, 30 ng/mL of bFGF, and 20% of knockout serum replacement all provided by Life Technologies. Typical hiPSC colonies formed on feeder cells were transferred to matrigel (BD-Biosciences) coated plates and expanded in Essential 8 Medium (Life Technologies) supplemented with 100 ug/mL of normocin (Invivogen). hiPSCs displayed embryonic stem cell-like morphology, expressed pluripotency markers (NANOG, OCT3, OCT4), and displayed trilineage differentiation potential after embryoid body differentiation and in vivo teratoma formation (see Supplementary Figure 1

Derivation of MSC-Like Cells from iPS-SHED and
iPS-FIB. iPS-SHED and iPS-FIB colonies from confluent plates were detached with accutase (Life Technologies). hiPSC colonies were partially dissociated via manual pipetting and the cells were plated onto matrigel-coated tissue culture dishes at 1 × 10 4 cells/cm 2 in MSC differentiation culture medium (Dulbecco's modified Eagle medium High Glucose-DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% nonessential amino acids, and 5 ng/mL of bFGF) for 14 days with media changes every 3 days. For subsequent passages, single-cell suspensions were prepared using TrypLE reagent (Life Technologies) and cells were passaged with a 1 : 3 split ratio in standard culture flasks (Corning) without matrigel coating.

Real-Time Quantitative PCR.
Total RNA was obtained from cell populations with the use of Nucleospin RNA II extraction kit (Macherey-Nagel) following manufacturer's recommendations. Briefly, one microgram of total RNA was converted into cDNA using Superscript II (Life Technologies), according to the manufacturer's recommendations. Real-time quantitative PCR reactions were performed with 2x SYBR Green PCR Master Mix (Life Technologies) and 25 nM-200 nM of each primer. Fluorescence was detected using ABI Prism 7500 Sequence Detection System, under standard temperature protocol. Primer pairs were designed with Primer-BLAST (http://www.ncbi.nlm .nih.gov/tools/primer-blast/; primer sequences are listed in Table 1, and their amplification efficiencies ( ) were determined by serial cDNA dilutions log 10 -plotted against Ct values, in which = 10 −1/slope . Expression of target genes was assessed relative to a calibrator cDNA (ΔCt). Finally, GeNorm v3.4 [17] was used to determine the most stable endogenous controls (among ACTB, TBP, and HMBS) and calculate normalization factors for each sample. The final expression values were determined based on a previous method [18], dividing −ΔCt by the corresponding normalization factor.

In Vitro Osteogenic Induction.
For osteogenic induction, MSC-like cells from iPS-SHED and from iPS-FIB were plated in 12-well plates (4 × 10 4 cells per well) and after   Figure 2). Next, we assessed the in vitro osteogenic potential of MSC-like cells from iPS-SHED, MSC-like cells from iPS-FIB and SHED during early in vitro osteogenesis by quantifying gene expression of key osteogenesis markers (DLX5 and RUNX2, two early transcription factors associated with osteogenesis, ALP and COL1A1, two early osteoblast markers, and BGLAP, a late osteoblast marker). ALP gene expression was upregulated in all cellular populations from day 2 to day 6 but showed higher expression in days 4 and 6 in MSClike cells from iPS-SHED and from iPS-FIB in comparison with SHED ( < 0.001). DLX5 peaked at day 2 in MSC-like cells from iPS-SHED and from iPS-FIB and was upregulated in SHED at all time points ( < 0.001). RUNX2 was also upregulated in SHED until day 6 of osteogenic induction, in comparison with MSC-like cells from iPS-SHED and from iPS-FIB ( < 0.001). COL1A1 was upregulated in MSClike cells from iPS-SHED and in SHED from day 2 to day 6 ( < 0.001) but showed no significant upregulation in MSClike cells from iPS-FIB during this period. BGLAP was not upregulated during this early stage of osteoinduction in any cellular population, as expected for a late osteoblast marker (Figure 3(a)).

Results
ALP enzymatic activity was higher in MSC-like cells from iPS-SHED when compared with MSC-like cells from iPS-FIB (2.3-fold increase, < 0.01) and with SHED (2.54-fold increase, < 0.001) after 9 days of in vitro osteoinduction (Figure 3(b)). Alizarin red S staining revealed more matrix mineralization in MSC-like cells from iPS-SHED when compared with SHED (4.36-fold increase, ( Figure 3(c)). In this time point, MSC-like cells from iPS-FIB showed a 2.99-fold increase ( < 0.001) in mineralized matrix production when compared with SHED. These data were validated by von Kossa staining after 14 and 21 days of in vitro osteogenesis (Figure 3(e)). Finally, we compared the expression of CD105 mRNAs between SHED, MSC-like cells from iPS-SHED and from iPS-FIB and found a lower expression of this gene in SHED when compared with the latter cellular populations ( < 0.001, Figure 3(d)).

Discussion
iPSC technology has gained attention to engender cellular populations to be used in tissue engineering, displaying selfrenewal, pluripotency, and differentiation plasticity similar to embryonic stem cells. Furthermore, the use of hiPSCs is not hindered by the ethical issues associated with the use of human embryos and permits the generation of therapeutically relevant cell types genetically compatible to patients, evading rejection drawbacks that may follow transplantation of nonautologous cells [19].  There is an increasing interest in investigating iPSCs for bone regenerative therapies and a series of studies have generated murine iPSCs and assessed their direct differentiation towards osteoblasts [20][21][22][23]. From a safety point of view, the use of progenitor cells instead of undifferentiated iPSCs for therapeutic purposes is advantageous since progenitor cells are already primed for a specific differentiation pathway and tumor formation risk is reduced [24]. Moreover, recent reports suggest that some of the reparative effects associated with MSC transplantation are not mediated by cellular differentiation per se but by paracrine factors secreted by them [25]; Fanganiello et al., submitted. The MSC differentiation from hiPSCs seemed to be successful as both MSC-like cells from iPS-SHED and from iPS-FIB displayed typical mesenchymal cell morphology, downregulation of pluripotency markers and similar cell surface antigen profiles and multipotential when compared with SHED. After in vitro osteoinduction, upregulation of osteogenesis markers DLX5 and RUNX2 in SHED in comparison with MSC-like cells from iPS-SHED and from iPS-FIB may indicate a previous commitment of this cell population towards the osteogenic lineage. However, in days 4 and 6 of osteoinduction, MSC-like cells from iPS-SHED and from iPS-FIB presented upregulation of ALP, a metalloenzyme known as a key early marker of osteogenesis. MSC-like cells from iPS-SHED also had more ALP enzymatic activity when compared with MSC-like cells from iPS-FIB and with SHED in midstage osteogenesis. MSC-like cells from iPS-SHED and from iPS-FIB produced significantly more mineralized extracellular matrix when compared with SHED. Overall, MSC-like cells from iPS-SHED were able to undergo induced in vitro osteogenesis in a more efficient fashion than MSCs from iPS-FIB or from the originating SHED populations.
One of the factors that could explain the higher efficiency of the in vitro osteogenesis in MSC-like cells from iPS-SHED and iPS-FIB in comparison with SHED might be related to 8 Stem Cells International the presence of a more homogeneous cellular population attributed to the direct plating protocol adopted. We have decided to choose the iPSC direct plating method over the embryoid body (EB) protocol since EBs are known to contain a heterogeneous mixture of cells with different degrees of multipotency that may limit their net osteogenic potential [26][27][28]. Accordingly, enhanced osteogenic differentiation has been associated with direct plating [29][30][31][32], and this method has been proposed to yield uniform batches of osteoprogenitor cells [31].
We also tested if the difference in osteogenic potential between the MSC-like cells from iPS-SHED from iPS-FIB and SHED is related to CD105 expression, as its lower expression has been associated with a higher osteogenic potential in MSCs harvested from human adipose tissue (hASCs) when compared with MSCs with higher CD105 expression [15]. Interestingly we found CD105 expression to be significantly lower in SHED when compared with both MSC-like cells from iPS-SHED and from iPS-FIB. Therefore, the higher osteogenic potential in this case may be due to other factors.
The difference in osteogenic potential here reported between MSC-like cells from iPS-SHED and from-iPS-FIB may possibly be related to a somatic epigenetic memory of the tissue of origin [33]. Derivation of pure populations of functionally differentiated cells from iPSCs is still challenging and different cell types show variable susceptibility to reprogramming. In fact, MSCs derived from iPSC lines from different tissues have been shown to exhibit variability in their differentiation profiles. Hynes et al. 2014 reported that MSC-like cells from iPSCs generated from periodontal ligament displayed higher osteogenic capacity both in vitro and in vivo when compared to MSC-like cells from iPSCs generated from lung and gingival fibroblasts, which was attributed to epigenetic memory of the donor tissue [34]. In another study, Sanchez-Freire et al. 2014 reported higher cardiac differentiation efficiency in MSC-like cells derived from iPSCs generated from cardiac progenitors in comparison with dermal fibroblasts from the same donor, which was demonstrated to be due to the retention of residual methylation signatures of the tissue of origin [35].

Conclusions
Our findings provide an important argument towards the use of iPSCs in tissue bioengineering since MSC-like cells from iPS-SHED and from iPS-FIB displayed higher osteogenic potential than SHED. We also suggest that cellular homogeneity and tissue of origin are important factors to be considered when planning to use iPSCs in bone regenerative medicine. CD105 does not seem to be a main factor involved in these differences. The dissection of the molecular basis of osteogenic differentiation in MSC-like cells from iPSCderived cells may furnish insights into the clinical usefulness of iPSCs from different sources.