Y42 - Assessing Neuronal Differentiation Efficiency of Mesenchymal Stem Cells from Multiple Tissue Sources

Introduction: : Each year, approximately 1.7 million people in the United States suffer traumatic brain injuries (TBIs), which can lead to hospitalization, long term neurological effects, or death. When a patient experiences a TBI, the injury occurs in two phases. The initial injury results from a force impacting the head. The secondary injury occurs in response and includes damage such as inflammation, gene activation, and cytotoxicity. The secondary response can lead to neurological disorders such as Alzheimer’s disease or dementia. Because the secondary response can proceed for hours to months, treatments like stem cell based therapy, can be administered before inflammation causes neurodegeneration. Human mesenchymal stem cells (hMSCs) may help treat TBI due to their anti-inflammatory properties and nerve regeneration abilities.
HMSCs are multipotent cells that can differentiate into several cell lineages including neurons, astrocytes, and microglia. HMSCs are derived from many sources including bone marrow (BM), umbilical cord (UC), and adipose tissue (AD), and have been explored in TBIs therapies. The purpose of this project is to show that neurons can be produced from multiple sources of hMSCs, including BM-hMSCs, UC-hMSCs, and AD-hMSCs. We hypothesize that neuronal differentiation efficiency will differ based on hMSC tissue origin and the growth media used. This is based on prior findings that suggest variations in hMSC differentiation potential depends on media composition and cell source. We assess morphological changes using immunostaining, gene expression using RT-qPCR, and electrical properties using dielectrophoresis (a label-free cell analysis technique), comparing differentiated cells to their undifferentiated counterparts.

Materials and

Methods: : Cell Culture
BM-hMSCs, UC-hMSCs, and AD-hMSCs were obtained from the American Type Culture Collection (ATCC). Cells were cultured in two media: (1) mesenchymal stem cell basal medium supplemented with a low serum growth kit from ATCC and (2) mesenchymal stem cell growth medium from PromoCell. Cells were maintained at 37°C and 5% CO₂, with media changes every 2–3 days, passaged at 80% confluency.

Neuronal Differentiation
Cells were seeded at 5500 cells/cm2 in 48-well plates and allowed to adhere for 2 days. Neuronal differentiation was induced using PromoCell neuronal differentiation media for 7 days, with media changes every 2 days. Protocol 1 refers to cells cultured in ATCC growth media and differentiated with PromoCell media. Protocol 2 refers to cells cultured and differentiated using PromoCell media.

Neuronal Assessments
Neuronal differentiation was assessed by analyzing morphology, gene expression, and electrical properties in differentiated versus undifferentiated cells.
Morphological Analysis
Cells were fixed and immunostained for MAP2 and NeuN neuronal markers. Images were captured at 20X magnification. Average aspect ratio was measured using FIJI to assess neurite outgrowth.
Gene Expression Analysis
RNA was extracted using the TRIzol Plus RNA Purification Kit (ThermoFisher), and cDNA was synthesized using SuperScriptTM IV VILOTM Master Mix (ThermoFisher). RT-qPCR was performed on QuantStudio 3 and 5 using NEUROD, GAPDH, and TBP.
Dielectrophoresis (DEP) Analysis
Electrical properties were measured using the 3DEP system (LabTech). Cells were suspended in 8.5% sucrose and 0.3% glucose buffer (conductivity 100 μS/cm). Membrane capacitance, cytoplasm conductivity, and transient slope were evaluated.

Results, Conclusions, and Discussions:: BM-hMSCs differentiated using Protocol 1 showed the greatest change in aspect ratio compared to undifferentiated control cells. They also had the highest percentage of MAP2-positive staining. AD-hMSCs differentiated using Protocol 1 had the highest percentage of NeuN-positive staining. UC-hMSCs showed an increase in MAP2 expression for both protocols but lower than what was observed for BM-hMSCs and AD-hMSCs. These results may be influenced by the initial morphology of hMSCs before differentiation. BM-hMSCs have a more fusiform morphology, which may allow them to adopt a neuron-like morphology more quickly than AD-hMSCs and UC-hMSCs. Also, AD-hMSCs and UC-hMSCs tend to have a more spherical morphology. Using dielectrophoresis, we observed no change in membrane capacitance but a decrease in transient slope for BM-hMSCs under both protocols. In contrast, AD-hMSCs showed a decrease in membrane capacitance with Protocol 1 and an increase with Protocol 2. Similarly, transient slope decreased with Protocol 1 and increased with Protocol 2 for AD-hMSCs. Trends in cytoplasm conductivity were variable, and UC-hMSCs are still being assessed. In the literature, dielectrophoresis has been used to distinguish fate potential in neural stem and progenitor cells (e.g., neuron-biased vs. astrocyte-biased) and to detect heterogeneity across hMSC sources. This supports its use here as a tool to assess neuronal differentiation.
In conclusion, BM-hMSCs differentiated using Protocol 1 generated neuron-like cells most efficiently, based on observed changes in morphology and protein expression during differentiation. In future work, we plan to use dielectrophoresis-based microfluidic cell sorting platforms to enrich subpopulations of hMSCs with greater potential for neuronal differentiation, with the goal of improving both the efficiency and consistency of the process. This approach may also help identify the most suitable hMSC source for neuronal applications, ultimately enhancing therapeutic strategies for TBIs and advancing tissue engineering efforts involving hMSCs.

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