Int J Stem Cells.  2019 Nov;12(3):449-456. 10.15283/ijsc18087.

Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

• Jain M
• Minocha E
• Tripathy N
• Singh N
• Chaturvedi CP
• Nityanand

• KMID: 2465885
• DOI: http://doi.org/10.15283/ijsc18087

Abstract

BACKGROUND AND OBJECTIVES
Most studies in cardiac regeneration have explored bone marrow mesenchymal stem cells (BM-MSC) with variable therapeutic effects. Amniotic fluid MSC (AF-MSC) having extended self-renewal and multi-potent properties may be superior to bone marrow MSC (BM-MSC). However, a comparison of their cardiomyogenic potency has not been studied yet.
METHODS
The 5-azacytidine (5-aza) treated AF-MSC and BM-MSC were evaluated for the expression of GATA-4, Nkx2.5 and ISL-1 transcripts and proteins by quantitative RT-PCR and Western blotting, respectively as well as for the expression of cardiomyogenic differentiation markers cardiac troponin-T (cTNT), beta myosin heavy chain (βMHC) and alpha sarcomeric actinin (ASA) by immunocytochemistry.
RESULTS
The AF-MSC as compared to BM-MSC had significantly higher expression of GATA-4 (183.06±29.85 vs. 9.80±0.05; p<0.01), Nkx2.5 (8.3±1.4 vs. 1.82±0.32; p<0.05), and ISL-1 (39.59±4.05 vs. 4.36±0.39; p<0.01) genes as well as GATA-4 (2.01±0.5 vs. 0.6±0.1; p<0.05), NKx2.5 (1.9±0.14 vs. 0.8±0.2; p<0.01) and ISL-1 (1.7±0.3 vs. 0.9±0.1; p<0.05) proteins. The AF-MSC also had significantly elevated expression of cTNT (5.0×10⁴±0.6×10⁴ vs. 3.5 ×10⁴±0.8×10⁴; p<0.01), β-MHC (15.7×10⁴±0.9×10⁴ vs. 8.2×10⁴±0.6×10⁴; p<0.01) and ASA (18.6×10⁴±4.9×10⁴ vs. 13.1×10⁴±3.0×10⁴; p<0.05) than BM-MSC.
CONCLUSIONS
Our data suggest that AF-MSC have greater cardiomyogenic potency than BM-MSC, and thus may be a better source of MSC for therapeutic applications in cardiac regenerative medicine.

Keyword

Amniotic fluid mesenchymal stem cells; Bone marrow mesenchymal stem cells; Cardiomyogenic potency; Cardiac transcription factors; Cardiac structural markers

MeSH Terms

Actinin
Amniotic Fluid*
Antigens, Differentiation
Azacitidine
Blotting, Western
Bone Marrow*
Female
Humans*
Immunohistochemistry
Mesenchymal Stromal Cells*
Regeneration
Regenerative Medicine
Therapeutic Uses
Troponin T
Ventricular Myosins
Actinin
Antigens, Differentiation
Azacitidine
Therapeutic Uses
Troponin T
Ventricular Myosins

Figure

• 

  Fig. 1 Morphological and characterization of AF-MSC and BM-MSC. (a) Representative photomicrographs (10X, 10 micron) of AF-MSC and BM-MSC showing a trigonal and fibroblastoid morphology respectively in 3rd passage; (b, c) Proliferation rate and Population Doubling Time (PDT) of the both AF-MSC and BM-MSC at different hours; (d) Representative flow cytometric histogram of AF-MSC and BM-MSC showing the presence of MSC markers (CD73, CD90 and CD105) & absence of hematopoietic stem cell markers (CD34, CD45 and HLA-DR). Values expressed as Mean±SD; **p<0.01.

• 

  Fig. 2 Adipogenic, osteogenic and chondrogenic differentiation of AF-MSC and BM-MSC. Representative photomicrographs showing the differentiation of AF-MSC and BM-MSC into (a) adipocytes, as demonstrated with Oil red O Staining and their relative intensity, (b) osteocytes, as demonstrated with Alizarin red staining and their relative intensity, (c) chondrocytes, as demonstrated with Alcian blue staining and their relative intensity. Control AF-MSC and BM-MSC (untreated cells) were negative for Oil red O, Alizarin red and Alcian Blue staining, respectively. Values expressed as Mean±SD; **p<0.01, ***p<0.001, *p<0.05 respectively.

• 

  Fig. 3 Expression of genes and proteins of cardiac transcription factors in AF-MSC and BM-MSC. (a) Representative reverse-transcription polymerase chain reaction photomicrographs showing expression of Cardiac transcription factors in 5-azacytitdine treated and untreated (control) AF-MSC and BM-MSC for GATA-4, NKx2.5 and ISL-1. Values expressed as Mean±SD; **p<0.01, *p<0.05, **p<0.01 respectively. (b) Representative immune-blots showing expression of the GATA-4, NKx2.5 and ISL-1 in both control and 5-aza treated in AF-MSC and BM-MSC (c) their relative intensity applied for comparison of relative protein expression. Values expressed as Mean±SD; *p<0.05, **p<0.01, *p<0.05 between differentiated vs. control respectively.

• 

  Fig. 4 Expression of cardiac structural markers in AF-MSC and BM-MSC. (a) Representative immunofluorescence photomicrographs (40x, 10 micron) of 5 aza treated AF-MSC and BM-MSC staining of cTNT, βMHC, and ASA proteins; (b) their relative intensity was applied for comparison of relative protein expression. Values expressed as Mean±SD; **p<0.01; **p<0.01, *p<0.05 between differentiated vs. control respectively. CTCF (Corrected Total Cell Fluorescence)=Integrated Density−(Area of selected cell×Mean fluorescence of background readings).

Reference

References

1. Duran JM, Makarewich CA, Sharp TE, Starosta T, Zhu F, Hoffman NE, Chiba Y, Madesh M, Berretta RM, Kubo H, Houser SR. Bone-derived stem cells repair the heart after myocardial infarction through transdifferentiation and paracrine signaling mechanisms. Circ Res. 2013; 113:539–552. DOI: 10.1161/CIRCRESAHA.113.301202. PMID: 23801066. PMCID: PMC3822430.

2. Karantalis V, Hare JM. Use of mesenchymal stem cells for therapy of cardiac disease. Circ Res. 2015; 116:1413–1430. DOI: 10.1161/CIRCRESAHA.116.303614. PMID: 25858066. PMCID: PMC4429294.

3. Elahi KC, Klein G, Avci-Adali M, Sievert KD, MacNeil S, Aicher WK. Human mesenchymal stromal cells from different sources diverge in their expression of cell surface proteins and display distinct differentiation patterns. Stem Cells Int. 2016; 2016:5646384. DOI: 10.1155/2016/5646384. PMID: 26770208. PMCID: PMC4684891.

4. Pandey AC, Lancaster JJ, Harris DT, Goldman S, Juneman E. Cellular therapeutics for heart failure: focus on mesenchymal stem cells. Stem Cells Int. 2017; 2017:9640108. DOI: 10.1155/2017/9640108. PMID: 29391871. PMCID: PMC5748110.

5. Afzal MR, Samanta A, Shah ZI, Jeevanantham V, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult bone marrow cell therapy for ischemic heart disease: evidence and insights from randomized controlled trials. Circ Res. 2015; 117:558–575. DOI: 10.1161/CIRCRESAHA.114.304792. PMID: 26160853. PMCID: PMC4553075.

6. Martin-Rendon E, Sweeney D, Lu F, Girdlestone J, Navarrete C, Watt SM. 5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies. Vox Sang. 2008; 95:137–148. DOI: 10.1111/j.1423-0410.2008.01076.x. PMID: 18557828.

7. Stolzing A, Jones E, McGonagle D, Scutt A. Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev. 2008; 129:163–173. DOI: 10.1016/j.mad.2007.12.002. PMID: 18241911.

8. Fan M, Chen W, Liu W, Du GQ, Jiang SL, Tian WC, Sun L, Li RK, Tian H. The effect of age on the efficacy of human mesenchymal stem cell transplantation after a myocardial infarction. Rejuvenation Res. 2010; 13:429–438. DOI: 10.1089/rej.2009.0986. PMID: 20583954.

9. Loukogeorgakis SP, De Coppi P. Concise review: amniotic fluid stem cells: the known, the unknown, and potential regenerative medicine applications. Stem Cells. 2017; 35:1663–1673. DOI: 10.1002/stem.2553. PMID: 28009066.

10. Antonucci I, Stuppia L, Kaneko Y, Yu S, Tajiri N, Bae EC, Chheda SH, Weinbren NL, Borlongan CV. Amniotic fluid as a rich source of mesenchymal stromal cells for transplantation therapy. Cell Transplant. 2011; 20:789–795. DOI: 10.3727/096368910X539074. PMID: 21054947.

11. Savickiene J, Treigyte G, Baronaite S, Valiuliene G, Kaupinis A, Valius M, Arlauskiene A, Navakauskiene R. Human amniotic fluid mesenchymal stem cells from second- and third-trimester amniocentesis: differentiation potential, molecular signature, and proteome analysis. Stem Cells Int. 2015; 2015:319238. DOI: 10.1155/2015/319238. PMID: 26351462. PMCID: PMC4553339.

12. Roubelakis MG, Pappa KI, Bitsika V, Zagoura D, Vlahou A, Papadaki HA, Antsaklis A, Anagnou NP. Molecular and proteomic characterization of human mesenchymal stem cells derived from amniotic fluid: comparison to bone marrow mesenchymal stem cells. Stem Cells Dev. 2007; 16:931–952. DOI: 10.1089/scd.2007.0036. PMID: 18047393.

13. Zheng YB, Gao ZL, Xie C, Zhu HP, Peng L, Chen JH, Chong YT. Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study. Cell Biol Int. 2008; 32:1439–1448. DOI: 10.1016/j.cellbi.2008.08.015. PMID: 18782626.

14. Yan ZJ, Hu YQ, Zhang HT, Zhang P, Xiao ZY, Sun XL, Cai YQ, Hu CC, Xu RX. Comparison of the neural differentiation potential of human mesenchymal stem cells from amniotic fluid and adult bone marrow. Cell Mol Neurobiol. 2013; 33:465–475. DOI: 10.1007/s10571-013-9922-y. PMID: 23478940.

15. Mareschi K, Castiglia S, Sanavio F, Rustichelli D, Muraro M, Defedele D, Bergallo M, Fagioli F. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta. Exp Hematol. 2016; 44:138–150.e1. DOI: 10.1016/j.exphem.2015.10.009. PMID: 26577566.

16. Ramasamy TS, Velaithan V, Yeow Y, Sarkar FH. Stem cells derived from amniotic fluid: a potential pluripotent-like cell source for cellular therapy? Curr Stem Cell Res Ther. 2018; 13:252–264. DOI: 10.2174/1574888X13666180115093800. PMID: 29336267.

17. Markmee R, Aungsuchawan S, Narakornsak S, Tancharoen W, Bumrungkit K, Pangchaidee N, Pothacharoen P, Puaninta C. Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells. Mol Med Rep. 2017; 16:6068–6076. DOI: 10.3892/mmr.2017.7333. PMID: 28849052. PMCID: PMC5865810.

18. Tripathy NK, Rizvi SHM, Singh SP, Garikpati VNS, Nityanand S. Cardiomyogenic heterogeneity of clonal sub-populations of human bone marrow mesenchymal stem cells. J Stem Cells Regen Med. 2018; 14:27–33. PMID: 30018470. PMCID: PMC6043656.

19. Batsali AK, Pontikoglou C, Koutroulakis D, Pavlaki KI, Damianaki A, Mavroudi I, Alpantaki K, Kouvidi E, Kontakis G, Papadaki HA. Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton’s jelly and bone marrow-derived mesenchymal stem cells. Stem Cell Res Ther. 2017; 8:102. DOI: 10.1186/s13287-017-0555-9. PMID: 28446235. PMCID: PMC5406919.

20. Armiñán A, Gandía C, Bartual M, García-Verdugo JM, Lledó E, Mirabet V, Llop M, Barea J, Montero JA, Sepúlveda P. Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells Dev. 2009; 18:907–918. DOI: 10.1089/scd.2008.0292. PMID: 18983250.

21. Yi Q, Xu H, Yang K, Wang Y, Tan B, Tian J, Zhu J. Islet-1 induces the differentiation of mesenchymal stem cells into cardiomyocyte-like cells through the regulation of Gcn5 and DNMT-1. Mol Med Rep. 2017; 15:2511–2520. DOI: 10.3892/mmr.2017.6343. PMID: 28447752. PMCID: PMC5428324.

22. Genead R, Danielsson C, Andersson AB, Corbascio M, Franco-Cereceda A, Sylvén C, Grinnemo KH. Islet-1 cells are cardiac progenitors present during the entire lifespan: from the embryonic stage to adulthood. Stem Cells Dev. 2010; 19:1601–1615. DOI: 10.1089/scd.2009.0483. PMID: 20109033.