The differentiation potential of stem cells from different
origins: Osteogenic differentiation from iPSCs, ESCs, and BMSC-derived
mesenchymal stem cells.
Cellular context affects the way that cells respond to different
biomaterials. Although stem cells from different sources retain the
capacity to divide and differentiate endlessly, each source imprints
subtle changes to cells that affect their differentiation process. To
address how stem cells from different sources respond to the same
biomaterial, we used data generated from mesenchymal stem cells (MSCs)
under differentiation on osteoinductive scaffolds in perfusion
bioreactors (de Peppo et al., 2013). These MSCs arose from three
different origins: they were differentiated from human induced
pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs),
or they were directly acquired from human bone marrow (BM) (de Peppo et
al., 2013). All these different cells were induced to MSCs using
serum-supplemented medium for 7 days and then more 10-11 passages (de
Peppo et al., 2013). The main focus of that study was proving the
feasibility of using hiPSCs for generation of osteoblasts.
We used this dataset to more deeply analyze the transcriptomic
differences between osteoblast differentiation of different cells of
origin. For hiPSC, hESC and bone marrow cells, we compared gene
expression profiles of MSCs before and after differentiation towards
osteoblasts. In summary, using the same criteria (FC>2 and
adjusted p-value <0.05) we found 416, 869, and 341
differentially expressed genes respectively for hiPSCs, hESCs, and
BM-derived MSCs differentiated into osteogenic fate (Fig. S1). Analysis
of the common genes in transcriptomic level from all differentiation
experiments using osteoinductive scaffolds in perfusion bioreactors
showed an up regulation of an osteoblast specific profile as the most
significant cell signature (Fig. 3a and 3b, Table S4). Many of these
genes are involved in extracellular matrix organization, including
COL12A1, COL8A2, and COMP. It has been shown that COL12A1 is involved in
regulation of osteoblast differentiation and bone matrix formation (Izu
et al., 2011). This suggests that MSCs generated from different stem
cells can successfully be committed into osteogenic fate. Despite the
similarities between all induced osteoblasts, we found that
differentially expressed genes during osteogenic differentiation of ESC
derived and iPCS derived MSCs clustered together but were markedly
different from osteogenic differentiation with BM origin (Fig. S2).
To get a better understanding of osteogenic differentiation from
different cells of origin, we restricted our list to genes that were
exclusively upregulated in cells differentiated from hESC, hiPSC, or BM.
There were 549, 197, and 134 genes significantly upregulated
(FC>2 and p-values <0.05) from hESC-MSC,
hiPSC-MSC, and BM-MSC derived osteoblasts, respectively. Gene set
enrichment analysis showed genes involved in extracellular matrix
organization enriched significantly in iPSC-MSC and ESC-MSC derived
osteoblasts, but not in BM-MSC derived osteoblasts (Fig. 3c and 3d). In
comparing genes involved in extracellular matrix organization between
iPSCs and ESCs, we found higher number of genes with more significantp-value related to this biological term in iPSCs than ESCs.
Extracellular matrix as a local microenvironment of osteoblast has
significant role in cell motility, communication and shape by providing
an anchorage site for cells and storing and presenting cytokines for
cell growth (Rozario & DeSimone, 2010).
To provide a better understanding of the different differentiation
potentials of MSCs from each origin, we made a gene regulatory network
for genes exclusively involved in the differentiation of ESC-MSCs into
osteoblasts. We then mapped gene expression profiles of osteoblasts
generated from all three cells of origin onto the ESC-MSC constructed
gene regulatory network (Fig. 3e, 3f, and 3g). Our results clearly
indicated these three cells of origin showed significantly different
responses in the gene expression level upon osteogenic differentiation.
For example, based on the gene regulatory network analysis, we found the
transcription factor MYC to be exclusively down-regulated in the ESC-MSC
osteogenic differentiation according to adjusted p-value of the
genes. MYC is a master regulator of cell proliferation and stemness, so
this suggests that the ESC-MSC derived osetoblasts were more
differentiated than the iPSC and BM derived MSCs. Additionally, 76% of
down-regulated genes in the ESC-MSC network are controlled by MYC,
further suggesting that the entire MYC pathway is dampened in the
ESC-MSC derived osteoblasts. Network based ontology revealed biological
processes related to cell cycle, mitochondrial and metabolism as most
significant processes (Fig. 3h).
Our results clearly showed that MSCs originated from different cells of
origin were transcriptionally differently respond to the osteogenic
differentiation. Despite the fact that all three stem cell populations
could differentiate into osteogenic lineage, it is evident that each
cell of origin resulted in a moderately distinct transcriptional profile
of the differentiated cell. Overall, through bioinformatic analysis of
biomaterial-specific responses, as well as analysis of cell-of-origin
based response, we highlight that both of these modalities are
instrumental in the optimization of targeted stem cell differentiation.