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. Methods and 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 β-catenin, decreased progressively during culture. Exposure to Wnt3a rescued β-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, α- and β-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.

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The optimal medium for cardiac differentiation of adult primitive cells remains to be established. We quantitatively compared the efficacy of IGF-1, dynorphin B, insulin, oxytocin, bFGF, and TGF-beta1 in inducing cardiomyogenic differentiation. Adult mouse skeletal muscle-derived Sca1+/CD45-/c-kit-/Thy-1+ (SM+) and Sca1-/CD45-/c-kit-/Thy-1+ (SM-) cells were cultured in basic medium (BM; DMEM, FBS, IGF-1, dynorphin B) alone and BM supplemented with insulin, oxytocin, bFGF, or TGF-beta1. Cardiac differentiation was evaluated by the expression of cardiac-specific markers at the mRNA (qRT-PCR) and protein (immunocytochemistry) levels. BM+TGF-beta1 upregulated mRNA expression of Nkx2.5 and GATA-4 after 4 days and Myl2 after 9 days. After 30 days, BM+TGF-beta1 induced the greatest extent of cardiac differentiation (by morphology and expression of cardiac markers) in SM- cells. We conclude that TGF-beta1 enhances cardiomyogenic differentiation in skeletal muscle-derived adult primitive cells. This strategy may be utilized to induce cardiac differentiation as well as to examine the cardiomyogenic potential of adult tissue-derived stem/progenitor cells.

Talimogene laherparepvec is a genetically modified herpes simplex virus type 1–based oncolytic immunotherapy for the local treatment of unresectable subcutaneous and nodal tumors in patients with melanoma recurrent after initial surgery. We report on two patients with melanoma who, after progression on numerous systemic therapies, derived clinical benefit from talimogene laherparepvec in an expanded-access protocol (ClinicalTrials.gov, NCT02147951). Intralesional talimogene laherparepvec (day 1, ≤4 ml 106 PFU/ml; after 3 weeks, ≤4 ml 108 PFU/ml every 2 weeks) was administered until complete response, no injectable tumors, progressive disease, or intolerance occurred. Patient 1 was 71 years old, had stage IIIB disease, and had previously received granulocyte–macrophage colony-stimulating factor, vemurafenib, metformin, ipilimumab, dabrafenib, trametinib, and pembrolizumab. Patient 2 was 45 years old, had stage IIIC disease, and had previously received nivolumab/ipilimumab combination therapy. There were marked reductions in the number and size of melanoma lesions during treatment with talimogene laherparepvec. Both patients experienced mild-to-moderate nausea and vomiting, which were managed using ondansetron, metoclopramide, and pantoprazole. Both patients completed treatment with talimogene laherparepvec in the expanded-access protocol on 24 November 2015, but received talimogene laherparepvec in clinical practice. Patient 1 continues to receive therapy (>60 weeks); patient 2 experienced a complete response at 23 weeks. Immunohistochemistry of a biopsied dermal metastasis from patient 1 showed a marked infiltration of CD4+ and CD8+ T cells after 1 year of treatment. Talimogene laherparepvec was active in patients with advanced melanoma with disease progression following multiple previous systemic therapies; no new safety signals were identified.

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