Noncanonical Wnt11 signaling is sufficient to induce cardiomyogenic differentiation in unfractionated bone marrow mononuclear cells.
BACKGROUND
Despite the frequent clinical use of adult unfractionated bone marrow mononuclear cells (BMMNCs) for cardiac repair, whether these cells are capable of undergoing cardiomyogenic differentiation in vitro remains uncertain. In addition, the role of Wnt signaling in cardiomyogenic differentiation of adult cells is unclear.


RESULTS
Unfractionated BMMNCs were isolated from adult mice via Ficoll-Paque density-gradient centrifugation and cultured in the presence of Wnt3a or Wnt11. In control BMMNCs, Wnt11 was not expressed, whereas the expression of markers of pluripotency (Oct-4 and Nanog), as well as that of Wnt3a and beta-catenin, decreased progressively during culture. Exposure to Wnt3a rescued beta-catenin expression and markedly increased the expression of Oct-4 and Nanog, concomitant with increased cell proliferation and CD45 expression. In contrast, exposure to ectopically expressed noncanonical Wnt11 markedly decreased the expression of Oct-4 and Nanog and induced mRNA expression (quantitative real-time reverse-transcription polymerase chain reaction) of cardiac-specific genes (Nkx2.5, GATA-4, atrial natriuretic peptide, alpha- and beta-myosin heavy chain, and cardiac troponin T) by day 3 with subsequent progression to a pattern characteristic of the cardiac fetal gene program. After 21 days, 27.6+/-0.6% and 29.6+/-1.4% of BMMNCs expressed the cardiac-specific antigens cardiac myosin heavy chain and cardiac troponin T, respectively (immunocytochemistry), indicating cardiomyogenic lineage commitment. Wnt11-induced cardiac-specific expression was completely abolished by the protein kinase C inhibitor bisindolylmaleimide I, partially abolished by the c-Jun-N-terminal kinase inhibitor SP600125, and attenuated by the Wnt inhibitor Dickkopf-1.


CONCLUSIONS
In adult density-gradient separated BMMNCs, canonical Wnt3a promotes stemness, proliferation, and hematopoietic commitment, whereas noncanonical signaling via Wnt11 induces robust cardiomyogenic differentiation in a protein kinase C- and c-Jun-N-terminal kinase-dependent manner.
Flaherty
Michael P
MP
Division of Cardiology, Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292, USA.
Abdel-Latif
Ahmed
A
Li
Qianhong
Q
Hunt
Greg
G
Ranjan
Smita
S
Ou
Qinghu
Q
Tang
Xian-Liang
XL
Johnson
Robin K
RK
Bolli
Roberto
R
Dawn
Buddhadeb
B
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D019070:Cell Lineage / Q000187:drug effects / Q000502:physiology; D002469:Cell Separation; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014162:Transfection; D019210:Troponin I / Q000235:genetics; D051153:Wnt Proteins / Q000235:genetics / Q000494:pharmacology; D051155:Wnt1 Protein / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000235:genetics
D019210:Troponin I; D051153:Wnt Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; C508957:Wnt11 protein, mouse; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D051176:beta Catenin; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases