12 / 16
Page 32
Insights in Enzyme Research
ISSN: 2573-4466
E u r o S c i C o n C o n g r e s s o n
Enzymology and
Molecular Biology
A u g u s t 1 3 - 1 4 , 2 0 1 8
P a r i s , F r a n c e
Enzymology 2018
Introduction:
Intervertebral disc (IVD) is closely related to low back pain, is the major cause of disability worldwide, with more
than 84% population experiencing pain in their life time. Biological and cell based therapies are on progress as an optional
treatment for IVD degeneration. Many studies have also revealed that mesenchymal stem cells (MSCs) can also be differentiated
into nucleus pulposus (NP) like cells phenotype. This study aimed to determine the newly defined healthy NP cells markers; HIF-
1α, HIF-2α, GLUT-1, Shh, Brachyury, Aggrecan, Collagen II, Carbonic anhydrase 3, Carbonic anhydrase 12, CD24, Cytokeratin 8,
Cytokeratin 18, and Cytokeratin 19 of Sprague-Dawley rat, whether these markers can be expressed in MSCs under co-culture
condition and could be identified the differentiation of MSCs into NP-like cells.
Methods:
NP cells and bone marrow derived MSCs from Sprague-Dawley rats were cultured under normoxic medium at 21% O
2
and 5% CO
2
at 37˚C and hypoxic medium at 2% O
2
, 5% CO
2
, 93% N
2
at 37˚C and MSCs were co-cultured with NP cells supernatant
with the concentration of 50% and 100% for 7 days under both normoxic and hypoxic medium. Differentiation of MSCs and
expression of recommended newly defined young healthy NP cells phenotypes were evaluated by quantitative real-time PCR
(qPCR), Western blotting and immunofluorescence staining microscopy. The results were determined among the groups using
unpaired Student’s t-test. p-values<0.05 considered significant.
Results:
MSCs co-cultured with the concentration of 50% NPcs supernatant; only collagen II showed the increased expression
while with the 100% NPcs supernatant; brachyury, collagen II, Glut-1, KRT18 and KRT19 showed higher expressions under
normoxic condition compared to MSC control. Under the hypoxic condition, MSCs co-cultured with 50% NPcs supernatant, HIF-
2α, Glut-1, aggrecan, collagen II, shh, KRT8, KRT19, CA3, CA12 and CD24 showed increased expression compared to MSC control.
More importantly, MSCs co-cultured with 100% NPcs supernatant under hypoxic condition, HIF-1α, HIF-2α, Glut-1, aggrecan,
collagen II, shh, brachyury, KRT8, KRT19, CA3, CA12 and CD24 showed upregulated increased expressions compared to the MSC
control, which showed that NP cells can stimulate MSCs differentiation to NP-like cells with paracrine interaction between MSCs
and NPcs under co-culture condition.
Conclusion:
This study suggested that MSCs were successfully differentiated into NP-like cells, which may be used as an ultimate
cell-based therapy for IVD regeneration.
Fig.
1: Relative protein expression by Western blot in MSCs co-cultured with NPcs supernatant (50% and 100% concentration) under
normoxia and hypoxia conditions for 7 days. Protein expression for each samples were normalized with housekeeping gene β-actin
Differentiation of mesenchymal stem cells into nucleus pulposus
like cells induced by co-culture system and hypoxia
Arjun Sinkemani
1
, Feng Wang
1
, Zhi-Yang Xie
1
, Lu Chen
1
, Cong
Zhang
1
and Xiao-Tao Wu
1
Zhongda Hospital, Southeast University, Nanjing, China
Arjun Sinkemani et al., Insights Enzyme Res 2018, Volume 2
DOI: 10.21767/2573-4466-C1-002