Differentiation of Mouse Induced Pluripotent Stem Cells (iPSCs) into Nucleus Pulposus-Like Cells in vitro
1
Jing L; 1Christoforou N; 1Leong KW; 1,2Setton LA and +2Chen J
Departments of Biomedical Engineering and 2Orthopaedic Surgery, Duke University, Durham, NC
[email protected]
formed EB-like cell clusters (Figure 2A, Bottom) similar to embryonic
INTRODUCTION: Autologous or allogeneic cell delivery to the
stem cells. Expression of NP markers during spontaneous
herniated or degenerated intervertebral disc (IVD) may promote tissue
1-4
differentiation. Undifferentiated iPSCs and EBs expressed many
regeneration and arrest degeneration . Induced pluripotent stem cells
integrin (α3, α5, α6 and β1 subunit) proteins and other NP markers
(iPSCs) derived from the patient’s somatic cells represent an attractive
(CD24 and CD54) on the cell surface, but not integrin β4 subunit and
cell source, having the potential to differentiate into various cell types5.
CD31 and CD90 (Table 1). Interestingly, higher % of cells expressed
To date, no studies have demonstrated that iPSCs can differentiate into
CD24 (a NP cell marker), CD31 and CD90 (mesenchymal stem cell
nucleus pulposus (NP) cells. We have previously found that human
markers) in EBs as compared to undifferentiated cells (Table 1). In
umbilical cord mesenchymal stromal cells can differentiate into NP-like
addition, cells in EBs expressed significant higher levels of mRNA for
cells in a laminin-rich pseudo-3D culture system6. The goal of this study
NOTO, FOXa2 and SHH (notochordal genes) as compared to
is to evaluate whether mouse iPSCs cultured in this same laminin-rich
undifferentiated cells (Figure 2B). Differentiation into NP-like cell
environment can also express a unique NP-like cell phenotype.
phenotypes in a 3D Matrigel culture system. Once cultured under
METHODS: MATERIALS AND METHODS. Cell generation and
differentiation conditions, iPSCs were shown to adopt a cell clustering
characterization. iPSCs were generated from primary mouse embryonic
morphology and to express some NP-related matrix proteins (LM511
fibroblasts (PMEF) through transient inducible over-expression of
and proteoglycans as indicated by Saf O, Figure 3), as well as many NP
transcription factors (OCT4, SOX2, KLF4 and MYC) by a lentiviralmarkers (cytokeratin 8, integrin subunit α3, α6 and β4, Figure 3);
based gene delivery system (a poly-cystronic vector with the Reverse
notably, expression of type II collagen
Tetracycline Transactivator (M2rtTA) and doxycycline). A previous
and vimentin were absent (Figure 3).
study confirmed pluripotent marker (OCT4, SOX2, NANOG and SSEA1) expression in the derived iPSC colonies and found that the derived
iPSCs were able to differentiate into different cell lineages including
cardiomyocytes7. Cell differentiation. Undifferentiated iPSCs were
seed on a PMEF feeder layer in proliferation medium (DMEM, 20%
FBS and LIF, 1000U/ml) and passaged twice (P2). Undifferentiated
cells were formed into 3D embryoid bodies (EBs) in ultra-low
attachment dishes (Corning) with proliferation medium w/o LIF for 3
days, and EBs were seeded (106/well, TranswellTM) on wells pre-coated
with Matrigel, then cultured in differentiation media (DMEM/F12+10%
Figure 2. A. Undifferentiated iPSCs cultured on a feeder layer (top) and
FBS) containing 2.5% Matrigel to provide for a pseudo-3D culture
formed EBs during spontaneous differentiation (bar = 100µm). B.
system6. NP markers and laminin (LM511) expression were evaluated
Relative mRNA levels for notochordal genes in EBs (3 days) normalized
in cells at three different stages (undifferentiated, EBs, day 7 post-NP
to undifferentiated iPSCs.
differentiation, as illustrated in Figure 1). Flow cytometry analysis.
Cells were recovered and incubated with the antibodies against CD24,
CD54, CD31, CD90, integrin subunits α3 (CD49c), α6 (CD49f), β1
(CD29) and β4 (CD104) with appropriate isotype controls and
fluorescently labeled secondary antibodies (Chemicon). Cells were
analyzed for fluorescence (Accuri C6) to quantify the percentage of cells
with positive surface proteins (%). RT-PCR. Undifferentiated cells and
EBs were harvested for total RNA isolation. Gene expression of
notochord-related transcriptional factors (NOTO, FOXa2 and SHH) was
analyzed via realtime RT-PCR (Bio-Rad) with β2-microglobulin as an
internal control. Immunohistochemistry. After 7 days post NPdifferentiation, cells were harvested for cryo-sectioning.
Cell
morphology and proteoglycan synthesis was assessed by histological
Figure 3. Immuno-staining for NP marker: type II collagen (Col II),
staining (H&E and Safranin O). Expression of NP markers was
proteoglycans (Saf O), vimentin (VIM), cytokeratin 8 (KRT8), laminin
evaluated for matrix proteins (type II collagen and LM511) and laminin
10/11 (LM511) and integrins (ITG α3, α6, β4) in differentiated iPSCs
related receptors (integrin subunits α3, α6, β1, β4) as well as other
cultured for 7 days in a 3D Matrigel system (bar = 50µm).
markers (vimentin and cytokeratin 8) by immunostaining.
DISCUSSION. The results of this study demonstrate that a laminin-rich
pseudo-3D culture environment promoted iPSC differentiation into a
cell type exhibiting some NP-like markers and morphology.
Extracellular matrix formation (proteoglycans and LM511) and the
presence of laminin receptors (integrin α3, β1, α6 and β4 subunit) and
cytokeratin 8 were detected in differentiated cells as early as day 7 of
NP-differentiation. These expression patterns of NP-like markers and
clustering morphology in differentiated cells were similar to that in
immature NP tissue in situ8,9. While no unique set of markers has been
Figure 1. Pseudo-3D culture system with Matrigel in Transwell inserts6
established for NP cells via consensus, these results are promising for
(adapted from Debnath et al. 2003).
the suggestion that iPSCs can be induced to differentiate into NP cells
that may be useful for NP regeneration.
Table 1. Flow cytometry analysis (% of positive cells) reveals that
undifferentiated iPSCs and spontaneously differentiated EBs express cell
SIGNIFICANCE. Our study evaluated a novel cell source and its
surface markers common in MSC or NP cells.
potential use for cellular therapy of intervertebral disc repair.
Markers
CD CD CD CD CD CD CD CD
REFERENCES. [1]Nishimura et al. Spine 1998,23:1531. [2]Okuma et al. J
24
54
49c 49f
29
104
31
90
Orthop Res 2000,18:988. [3]Meisel et al. Eur Spine J 2006,15(S3):397. [4]Sakai
Undifferentiated
17
54
22
27
81
1
4
0
Eur Spine J 2008,17:S452. [5]Yamanaka, Cell 2009,137:13. [6]Chon et al. Tans
1
(Day 0)
EB (day 3)
28
35
9
27
85
3
20
15
RESULTS: Cell morphology. iPSCs proliferated and formed colonies
on PMEF feeder layers (Figure 2A, Top). When cultured in ultra-low
attachment dishes without LIF for spontaneous differentiation, iPSCs
ORS 2011,36:600. [7]Christoforou et al. ISSCR 9th Annual Meeting 2011.
[8]Chen et al. Connect Tissue Res 2009,50:294. [9]Rufai et al. Anat Embryol
(Berl) 1995,192:53. [10]Debnath ea al. Methods. 2003,30:256.
ACKNOWLEDGMENTS. Supported by NIH R01AR057410,
R01EB002263, R01AR047442, R01HL083008, AOSpine Foundation
and FAMRI Young Clinical Scientist award (NC).
Paper No. 0031 • ORS 2012 Annual Meeting