Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold
Authors/Creators
Description
Highlights of the Study
This study investigated the potential of purmorphamine (PMA), a small-molecule activator of Sonic hedgehog (Shh) signaling, to promote the differentiation of human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) into motor neuron-like cells. By combining PMA-mediated biochemical induction with a three-dimensional electrospun polycaprolactone (PCL) nanofibrous scaffold, the study demonstrated enhanced cell viability and motor neuron differentiation compared with conventional two-dimensional culture, highlighting a promising strategy for neural tissue engineering and potential cell-based therapies for neurological disorders.
Key findings:
🟢 Small-molecule induction: Purmorphamine was successfully used as a small-molecule Shh signaling activator to induce differentiation of WJ-MSCs toward a motor neuron-like phenotype, providing an alternative to the Shh protein itself.
🟢 Nanofibrous scaffold fabrication: Electrospinning produced homogeneous, bead-free, and randomly oriented PCL nanofibers with an average fiber diameter of approximately 100 nm, forming a porous three-dimensional environment suitable for cell attachment and growth.
🟢 Cell–scaffold interaction: SEM observations demonstrated successful attachment, spreading, and growth of WJ-MSCs on the electrospun PCL nanofibrous scaffold.
🟢 Enhanced cell viability: MTT analysis showed significantly greater WJ-MSC viability on the PCL nanofibrous scaffold than on the two-dimensional tissue culture polystyrene (TCP) control at days 5 and 7, supporting the biocompatibility of the three-dimensional scaffold.
🟢 Motor neuron induction protocol: WJ-MSCs were exposed to a differentiation protocol incorporating 1 μM purmorphamine and retinoic acid, followed by BDNF treatment, resulting in motor neuron-like differentiation over a 15-day induction period.
🟢 Motor neuron protein expression: Immunofluorescence analysis after 15 days demonstrated expression of the motor neuron-associated markers NF-H, choline acetyltransferase (ChAT), and Islet-1 in differentiated WJ-MSCs.
🟢 Improved marker expression on PCL: Cells cultured on PCL showed higher expression of NF-H (50%), ChAT (78%), and Islet-1 (82%) compared with cells cultured on TCP, which exhibited approximately 42%, 61%, and 76%, respectively.
🟢 ChAT enhancement: Among the immunofluorescence results, ChAT expression was significantly higher in the PCL scaffold group than in the conventional TCP culture, supporting improved cholinergic motor neuron differentiation in the three-dimensional environment.
🟢 Gene-expression profile: qRT-PCR demonstrated significant increases in the expression of mature motor neuron-associated genes Islet-1, ChAT, NF-H, and HB9 following PMA-mediated differentiation.
🟢 Developmental marker regulation: The early neural markers nestin and Pax6 were downregulated following induction, while mature motor neuron-associated markers increased, indicating progression toward a more differentiated neuronal phenotype.
🟢 3D culture advantage: Expression of motor neuron-related genes was higher in cells cultured on the PCL nanofibrous scaffold than in the corresponding two-dimensional TCP culture, demonstrating the supportive role of the nanofibrous microenvironment.
🟢 Shh pathway activation: PMA promotes neural differentiation through activation of the Smoothened (SMO) receptor within the Hedgehog signaling pathway, which is associated with increased downstream Gli1 expression.
🟢 PMA as a Shh alternative: As a synthetic small molecule, PMA offers potential advantages over naturally occurring Shh protein, including greater stability while targeting the same signaling pathway involved in motor neuron development.
🟢 Synergistic differentiation strategy: Combining biochemical stimulation through PMA with the physical three-dimensional microenvironment provided by electrospun PCL enhanced motor neuron-like differentiation relative to conventional two-dimensional culture.
🟢 Regenerative medicine potential: The PMA/PCL strategy provides a potential in vitro platform for generating motor neuron-like cells from an accessible mesenchymal stem cell source and may contribute to future stem cell-based approaches for neurological disorders.
Overall, the study demonstrates that activation of the Shh/Hedgehog signaling pathway using purmorphamine, combined with a three-dimensional electrospun PCL nanofibrous scaffold, effectively promotes the differentiation of WJ-MSCs into motor neuron-like cells. The enhanced cell viability and increased expression of motor neuron-specific markers on the PCL scaffold emphasize the complementary roles of biochemical signaling and the nanofibrous microenvironment, providing a promising strategy for motor neuron generation and neural tissue engineering.
ARTICLE LINK
https://doi.org/10.1007/s12035-016-0090-1
DOI
10.1007/s12035-016-0090-1
HOW TO CITE
Bahrami, N.; Bayat, M.; Mohamadnia, A.; Khakbiz, M.; Yazdankhah, M.; Ai, J.; Ebrahimi-Barough, S. Purmorphamine as a Shh Signaling Activator Small Molecule Promotes Motor Neuron Differentiation of Mesenchymal Stem Cells Cultured on Nanofibrous PCL Scaffold. Molecular Neurobiology 2017, 54, 5668–5675. DOI: 10.1007/s12035-016-0090-1.
KEYWORDS
Motor neuron differentiation • Wharton’s jelly mesenchymal stem cells • Purmorphamine • PCL scaffold
Abstract
There is variety of stem cell sources but problems in ethical issues, contamination, and normal karyotype cause many limitations in obtaining and using these cells. The cells in Wharton’s jelly region of umbilical cord are abundant and available stem cells with low immunological incompatibility, which could be considered for cell replacement therapy. Small molecules have been presented as less expensive biologically active compounds that can regulate different developmental process. Purmorphamine (PMA) is a small molecule that, according to some studies, possesses certain differentiation effects. In this study, we investigated the effect of the PMA on Wharton’s jelly mesenchymal stem cell (WJ-MSC) differentiation into motor neuronal lineages instead of sonic hedgehog (Shh) on PCL scaffold. After exposing to induction media for 15 days, the cells were characterized for expression of motor neuron markers including PAX6, NF-H, Islet1, HB9, and choline acetyl transferase (ChAT) by quantitative reverse transcription (PCR) and immunocytochemistry. Our results demonstrated that induced WJ-MSCs with PMA could significantly express motor neuron markers in RNA and protein levels 15 days post induction. These results suggested that WJ-MSCs can differentiate to motor neuron-like cells with PMA on PCL scaffold and might provide a potential source in cell therapy
for nervous system.