Synergistic Effect of BDNF and FGF2 in Efficient Generation of Functional Dopaminergic Neurons from human Mesenchymal Stem Cells

Manisha Singh,Shantanu Chowdhary,Rinkey Sharma,Nitika Monga,Sujata Mohanty,Anupama Kakkar,Manish Kumar,O P Kharbanda,Balram Airan

doi:10.1038/s41598-017-11028-z

Abstract

To understand the process of neurogenesis, generation of functional dopaminergic (DAergic) neurons from human mesenchymal stem cells (hMSCs) is important. BDNF has been reported to be responsible for inducing neuronal maturation and functionality. Previously, we have reported the efficient generation of neurons from human bone marrow derived MSCs using FGF2 alone. We hypothesize that hMSCs from various tissues [(bone marrow (BM), adipose tissue (AD) and dental pulp (DP)], if treated with BDNF on 9th day of induction, alongwith FGF2 will generate functional DAergic neurons. Hence, cells were characterized at morphometric, transcription and translational levels for various markers like MAP2, TH, NGN2, PITX3, DAT, synaptophysin, Kv4.2 and SCN5A. Functionality of in vitro generated neurons was studied by calcium ion imaging. Result analysis depicted that BDNF has effect on expression of dopaminergic neuronal markers at gene and protein levels and functionality of neurons. Among these hMSCs, DP-MSC showed significantly better neuronal characteristics in terms of morphology, expression of neuronal markers and foremost, functionality of neurons. From the present study, therefore, we concluded that i) BDNF has additive effect on neuronal characteristics and functionality ii) DP-MSC are better MSC candidate to study DAergic neurogenesis and perform future studies.

Highlights

Over a period of time, several research groups have reported the in vitro differentiation of human Mesenchymal Stem Cells into neuronal cells, using various strategies like chemicals, growth factors, conditioned media, co- culture, direct genetic programming, differentiation from induced pluripotent stem cells and by using scaffolds to mimic the matrix[8,9,10,11,12,13,14,15,16,17]
Addition of BDNF to the induction medium is reported to increase the number of functional neurons[18] we aimed to evaluate and compare the differential effect of BDNF in inducing the functionality in DAergic neurons generated from human Mesenchymal Stem Cells (hMSCs)
Proliferation studies suggest that population doubling time of DP-MSC is lesser than that of Bone Marrow- Mesenchymal Stem Cells (BM-MSC) and Adipose tissue derived Mesenchymal Stem Cells (AD-MSC) seconding the observations of MTT assay that proliferation rate of DP-MSC is higher than that of other two hMSC counterparts (Supplementary Fig. 2)

Summary

Introduction

Over a period of time, several research groups have reported the in vitro differentiation of human Mesenchymal Stem Cells (hMSCs) into neuronal cells, using various strategies like chemicals, growth factors, conditioned media, co- culture, direct genetic programming, differentiation from induced pluripotent stem cells and by using scaffolds to mimic the matrix[8,9,10,11,12,13,14,15,16,17]. Understanding the genesis of DAergic neurons will help in devising drug testing cell models or stem cell based treatment regimes in future. Various growth factors such as FGF2, FGF8, SHH, BDNF and all- trans retinoic acid (ATRA) have been used widely to generate DAergic neurons fron MSC. The DAergic neurons derived from BM-MSC showed expression of DA- specific marker, tyrosine hydroxylase (TH) with all the induction cocktails. We have compared the response of hMSCs obtained from different human tissue sources (BM, AD and DP) towards the two protocols of dopaminergic neuronal induction

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Conclusion