Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Summary

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Abstract

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Introduction

Experimental and clinical evidence demonstrates that cell transplantation can moderately improve the left ventricle contractile performance of failing hearts^{1,2,3,4,5,6,7,8,9}. Recent advances have opened further appealing opportunities for cardiovascular regeneration; these include the forced expression of reprogramming factors in somatic cells to induce pluripotency an....

Protocol

1. Comet Assay

1. Reagent preparation
   1. For low-melting-point agarose, place 500 mg of low-melting agarose in 100 mL of DNA-/RNA-free water. Heat the bottle in a microwave oven until the agarose dissolves. Place the bottle in a 37 °C water bath until needed.
      NOTE: For further reading, see Azqueta et al., who studied the effects of agarose concentration on the DNA-unwinding time and several electrophoresis factors²³.
   2. Dilute SYBR green DNA dye in Tris-EDTA (TE) Buffer at a 1:10,000 ratio to obtain a 1x working stock dye solution. This dye is light-sensitive, so store it ....

Results

Human induced pluripotent stem cells were cultured, and the DNA damage and the tail moment, which were used as a measure of DNA integrity, were analyzed by comet assay. iPS cells were embedded in low-melting-point agarose and placed on a glass slide. The cells were, then, treated with lysis buffer, followed by an alkaline solution, to obtain supercoiled DNA. Nucleoids were electrophoresed and comets were visualized by DNA dye (Figure 1A-D). T.......

Discussion

DNA integrity portrays cell integrity. Cells with damaged DNA are frequently in stress and eventually lose their integrity. The integrity of stem and stem-cell-derived cells that are being propagated for the purpose of transplantation is principal for the cells to perform their desired function. Transplanting cells with damaged DNA would result in a poor engraftment rate and performance of the cell^8,20. Therefore, examining the DNA integrity prior to cell transpl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin	Corning	25200056
8-well chamber slide	Millicell	PEZGS0816
Accutase	Stemcell Technologies	7920	Cell detachment solution
Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG	Jackson Immuno Research Laboratories	711-545-152 (RRID: AB_2313584)
Bovine Serum Albumin	Sigma-Aldrich	A7906
Cy3 AffiniPure Donkey Anti-Rabbit IgG	Jackson Immuno Research Laboratories	711-165-152 (RRID:AB_2307443)
DAPI	Sigma-Aldrich	D9564
Gibco B-27 Supplement, Serum Free,	Gibco	17504-044
Matrigel growth factor reduced	Corning	354230
mTeSR1	Stemcell Technologies	85850
PhalloidinA488	Life Technology	A12379
Phospho-Histone H2A.X (Ser139) Antibody	Cell Signaling Technology	2577 (RRID: AB_2118010)
RPMI	Gibco	11875-093
SYBR Green I nucleic acid gel stain	Sigma-Aldrich	S9430
Triton X-100	Fisher scientific	BP151-100
UltraPure Low melting Point Agarose	Invitrogen	16520050
Vectashield	Vector Laboratories	H-1000	Antifade