Characterization of Genetically Engineered Mesenchymal Stem Cells for Neurotherapeutic Applications

Abstract

Source: Sharma, A. D., et.al. High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies. J. Vis. Exp. (2015)

This video demonstrates the process of characterizing transgenic mesenchymal stem cells engineered to express therapeutic neurotrophic factors. It outlines the steps involved in fixing, permeabilizing, and staining the cells with primary and secondary antibodies targeting a cell proliferation marker, followed by analysis using a high-content screening system to capture fluorescent signals in the nuclei. Increased proliferation marker expression confirms the suitability of the transgenic cells for neurotherapeutic applications.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Ki67 Cell Proliferation Assay

1. Ki67 Cell Proliferation Assay (Immunocytochemistry)
   1. Rinse the cell cultures with 0.1 M phosphate (PO₄) buffer for one minute two times. Fix the culture with 4% paraformaldehyde (PFA) for 20 min at room temperature. Remove the PFA and rinse the wells with PBS for seven minutes three times.
   2. After the final rinse, add 100 µl of blocker solution (phosphate buffer saline, 5% normal donkey serum, 0.4% bovine albumin serum, and 0.2% Triton X-100....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well plates	Greiner Bio One	655090	96 well plates selected for use in ImageXpress
Bovine serum albumin (BSA)	Sigma-Aldrich (St. Louis, MO)	A9647
Rabbit anti-Ki67 antibody	Abcam (Cambridge, MA)	Ab16667	1:200 dilution
DAPI	Invitrogen (Carlsbad, CA)	D3571
Donkey anti-Rabbit Cy3	Jackson Immuno Res Lab (West Grove, PA)	711-165-152	1:500 dilution
Mouse MSCs of adult C57BL/6 mice	Tulane Cent for Gene Therapy (New Orleans, LA)		Isolated from bone marrow
Genetically modified MSCs (GFP, BDNF, GDNF, BDNF/GDNF)	These cells were obtained from our previous study : Ye et. al. (in preparation)
Normal donkey serum (NDS)	Jackson Immuno Res Lab (West Grove, PA)	017-000-121
Paraformaldehyde (PFA)	Fisher Scientific (Hampton, NH)	O4042	4% PFA in 0.1M PO4 buffer
Phosphate buffered saline (PBS)	Sigma-Aldrich (St. Louis, MO)	P4417
Triton X-100	Sigma-Aldrich (St. Louis, MO)	X100
ImageXpress Micro	Molecular devices (Sunnyvale, CA)	ImageXpress micro	High content screening system
MetaXpress 4.0	Molecular devices (Sunnyvale, CA)	MetaXpress 4.0	Image acquisition and analysis software
KH₂PO₄	Fisher Scientific (Hampton, NH)	P285	For PO₄ buffer
K₂HPO₄	Fisher Scientific (Hampton, NH)	P288	For PO₄ buffer