Biochemical and Biophysical Research Communications
Neurotrophic factor mediated neuronal differentiation of human cord blood mesenchymal stem cells and their applicability to assess the developmental neurotoxicity
Introduction
Neurogenesis is defined as a process of generating functional neurons from their precursors [1]. Adult neurogenesis is a dynamic, fine-tuned process and subjected to modulation by various physiological, pathological and pharmacological stimuli. Earlier it was believed that damaged neurons could not be restored, although recently many developmental studies has built the way for restoration of neurogenesis [2]. Functional integration of new neurons in the adult central nervous system (CNS) was first shown in songbirds [1]. Multipotent neural stem cells were later derived from adult mammalian brain [3] which subsequently found to play the role of raw material for neurogenesis. In the mammalian brain, there are various growth factors/neurotrophins required for neurogenesis. Among them, nerve growth factor (NGF) is the leading growth factor which regulates neurogenesis process up to a great extent. NGF was discovered almost 5 decades back, the exact role of NGF was not clear but with the availability of tools that allow sensitive and specific measurements of RNA and protein levels for NGF, it has become evident that the role of NGF is beyond the cellular development and nerve cells regulation [4]. NGF has been found to indulge in the cellular activities as rescue from cell injury and promoting cell repair systems [5]. NGF has been employed to convert many candidate cells as PC12 [6], SH-SY5Y [7], rat neural stem cell [8] into mature neurons under in vitro conditions. The ability of NGF for neuronal development can be a boon in the restoration of neurodegenerative diseases.
Researchers are using many biological systems to study the developmental neuroprotection and neuro restoration. Mesenchymal stem cells (MSCs) comprise of a rare population of multipotent progenitors having ability of haematopoiesis as well as differentiation into various cell subtypes [9]. MSCs can be isolated from number of sources. The sources can be an adult tissue as bone marrow (BM), peripheral blood (PB) and adipose tissue (AT) or neonatal birth-associated tissues as placenta (PL), umbilical cord (UC) and cord blood (CB) [10]. Besides MSCs, other stem/progenitor cell populations from cord blood include hematopoietic stem cells (HSCs) and two endothelial populations such as endothelial progenitor cells (EPCs) and endothelial colony-forming cells (ECFCs). The amounts of MSCs which can be obtained by these isolations vary enormously. Pittenger et al. isolated MSCs from bone marrow by density gradient centrifugation to eliminate unwanted cell types and only 0.001–0.01% of the cells isolated from the density interface were MSCs [11]. These cells show positive expression of CD105 (SH2), CD73 (SH3), CD44 and CD90 and negative expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR on their surface [12].
As we have mentioned, MSCs can be differentiated into various lineages as osteogenic, adipogenic, chondrogenic and neuronal subtypes. These MSCs, for their qualities like multipotency, plasticity and easy accessibility, have attracted various cell based therapies. These therapies may be associated with either direct replacement of damaged cells by exogenously implantation of MSCs or endogenous regeneration. Countless data signified successful use of MSCs in haematology, cancer therapy and numerous acquired or inherited genetic disorders. The therapeutic potential of these cells have been displayed in experimental treatment of various neurological diseases and neural tissue injuries [13]. Researchers have identified the neurogenic potential of MSCs by using many chemicals and various growth factors [14]. But endogenous factors, which are present in brain were not studied to check neuronal induction capacity of MSCs. Since, neurogenesis process is supported by neurotrophin family in brain. So for restoration purpose, endogenous factors should be studied. Therefore, in the current study, we explored the neurogenic potential of human umbilical cord blood derived MSCs by using NGF.
Section snippets
Reagents and consumables
All the chemicals, reagents, and kits used in this study were purchased from Stem Cell Technologies and Sigma, unless otherwise stated. Recombinant human basic nerve growth factor (rhbFGF) was purchased from PeproTech. Alpha minimal essential medium (α MEM), MSC qualified FBS, antibiotic solution (100X), GlutaMAX™-I (100X), Sodium bicarbonate (7.5%), fluorescent antibodies, and D-PBS were purchased from Gibco (Invitrogen, Grand Island, NY, USA). Antibodies were purchased from Millipore (USA).
Isolation and characterization of stem cells from cord blood
Human umbilical Cord blood derived adherent stem cells were grown on standard plastic surfaces with a fibroblastoid appearance. It was found in western blotting that MSCs have expressions for CD90, CD73 and CD105 surface markers. SH-SY5Y was taken as negative control for surface markers since this cell line is of human origin. CD90 were present in MSCs as well as in SH-SY5Y, as CD90 was also reported in SH-SY5Y [17] (Fig. 1B). The results of surface markers were also confirmed by
Discussion
MSCs have been one of the pioneer stem cells utilized in clinical regenerative medicine with multipotent differentiation capacity and autologous transplantation feasibility [9]. Experimental and preclinical studies have given successful insight for the use of MSCs in curing variety of acute and slowly progressive diseases [19]. MSC seems to be promising tool for therapeutic applications in incurable neurological disorders; however precise mechanism of their protective action is still unclear.
Declaration of no conflict of interest
Authors of this manuscript have no conflict of interest among them or anybody else regarding the scientific contents, financial matters and otherwise.
Acknowledgement
The authors are grateful to the Director of Indian Institute of Toxicology Research, Lucknow India, for keen interest in the present work. University grant commission (UGC) New Delhi, is acknowledged for providing a fellowship to Ms. Sadaf Jahan.
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