ABSTRACT
OBJECTIVE: Myocardial infarctions constitute a major factor contributing to non-natural mortality world-wide. Clinical trials of myocardial regenerative therapy, currently pursued by cardiac surgeons, involve administration of stem cells into the hearts of patients suffering from myocardial infarctions. Unfortunately, surgical acquisition of these cells from bone marrow or heart is traumatic, retention of these cells to sites of therapeutic interventions is low, and directed differentiation of these cells in situ into cardiomyocytes is difficult. The specific aims of this work were: (1) to generate autologous, human, pluripotent, induced stem cells (ahiPSCs) from the peripheral blood of the patients suffering myocardial infarctions; (2) to bioengineer heterospecific antibodies (htAbs) and use them for recruitment of the ahiPSCs to infarcted myocardium; (3) to initiate in situ directed cardiomyogenesis of the ahiPSCs retained to infarcted myocardium. METHODS: Peripheral blood was drawn from six patients scheduled for heart transplants. Mononuclear cells were isolated and reprogrammed, with plasmids carrying six genes (NANOG, POU5F1, SOX2, KLF4, LIN28A, MYC), to yield the ahiPSCs. Cardiac tissues were excised from the injured hearts of the patients, who received transplants during orthotopic surgery. These tissues were used to prepare in vitro models of stem cell therapy of infarcted myocardium. The htAbs were bioengineered, which simultaneously targeted receptors displayed on pluripotent stem cells (SSEA-4, SSEA-3, TRA-1-60, TRA-1-81) and proteins of myocardial sarcomeres (myosin, α-actinin, actin, titin). They were used to bridge the ahiPSCs to the infarcted myocardium. The retained ahiPSCs were directed with bone morphogenetic proteins and nicotinamides to differentiate towards myocardial lineage. RESULTS: The patients' mononuclear cells were efficiently reprogrammed into the ahiPSCs. These ahiPSCs were administered to infarcted myocardium in in vitro models. They were recruited to and retained at the treated myocardium with higher efficacy and specificity, if were preceded with the htAbs, than with isotype antibodies or plain buffers. The retained cells differentiated into cardiomyocytes. CONCLUSIONS: The proof of concept has been attained, for reprogramming the patients' blood mononuclear cells (PBMCs) into the ahiPSCs, recruiting these cells to infarcted myocardium, and initiating their cardiomyogenesis. This novel strategy is ready to support the ongoing clinical trials aimed at regeneration of infarcted myocardium.
ABSTRACT
INTRODUCTION: Cancer of the testes is currently the most frequent neoplasm and a leading cause of morbidity in men 15-35 years of age. Its incidence is increasing. Embryonal carcinoma is its most malignant form, which either may be resistant or may develop resistance to therapies, which results in relapses. Cancer stem cells are hypothesized to be drivers of these phenomena. SPECIFIC AIM: The specific aim of this work was identification and isolation of spectra of single, living cancer stem cells, which were acquired directly from the patients' biopsies, followed by testing of their pluripotency. PATIENTS METHODS: Biopsies were obtained from the patients with the clinical and histological diagnoses of the primary, pure embryonal carcinomas of the testes. The magnetic and fluorescent antibodies were genetically engineered. The SSEA-4 and TRA-1-60 cell surface display was analyzed by multiphoton fluorescence spectroscopy (MPFS), flow cytometry (FCM), immunoblotting (IB), nuclear magnetic resonance spectroscopy (NMRS), energy dispersive x-ray spectroscopy (EDXS), and total reflection x-ray spectroscopy (TRXFS). The single, living cells were isolated by magnetic or fluorescent sorting followed by their clonal expansion. The OCT4A, SOX2, and NANOG genes' transcripts were analyzed by qRTPCR and the products by IB and MPFS. RESULTS: The clones of cells, with the strong surface display of TRA-1-60 and SSEA-4, were identified and isolated directly from the biopsies acquired from the patients diagnosed with the pure embryonal carcinomas of the testes. These cells demonstrated high levels of transcription and translation of the pluripotency genes: OCT4A, SOX2, and NANOG. They formed embryoid bodies, which differentiated into ectoderm, mesoderm, and endoderm. CONCLUSION: In the pure embryonal carcinomas of the testes, acquired directly from the patients, we identified, isolated with high viability and selectivity, and profiled the clones of the pluripotent stem cells. These results may help in explaining therapy-resistance and relapses of these neoplasms, as well as, in designing targeted, personalized therapy.
ABSTRACT
BACKGROUND: Ongoing clinical trials, in regenerative therapy of patients suffering from myocardial infarctions, rely primarily upon administration of bone marrow stem cells to the infarcted zones. Unfortunately, low retention of these cells, to the therapeutic delivery sites, reduces effectiveness of this strategy; thus it has been identified as the most critical problem for advancement of cardiac regenerative medicine. SPECIFIC AIMS: The specific aim of this work was three-fold: (1) to isolate highly viable populations of human, autologous CD34+, CD117+, and CD133+ bone marrow stem cells; (2) to bioengineer heterospecific, tetravalent antibodies and to use them for recruiting of the stem cells to regenerated zones of infarcted myocardium; (3) to direct vasculogenesis of the retained stem cells with the defined factors. PATIENTS METHODS: Cardiac tissue was biopsied from the hearts of the patients, who were receiving orthotopic heart transplants after multiple cardiac infarctions. This tissue was used to engineer fully human in vitro models of infarcted myocardium. Bone marrow was acquired from these patients. The marrow cells were sorted into populations of cells displaying CD34, CD117, and CD133. Heterospecific, tetravalent antibodies were bioengineered to bridge CD34, CD117, CD133 displayed on the stem cells with cardiac myosin of the infarcted myocardium. The sorted stem cells were administered to the infarcted myocardium in the in vitro models. RESULTS: Administration of the bioengineered, heterospecific antibodies preceding administration of the stem cells greatly improved the stem cells' recruitment and retention to the infarcted myocardium. Treatment of the retained stem cells with vascular endothelial growth factor and angiopoietin efficiently directed their differentiation into endothelial cells, which expressed vascular endothelial cadherin, platelet / endothelial cell adhesion molecule, claudin, and occludin, while forming tight and adherens junctions. CONCLUSIONS: This novel strategy improved retention of the patients' autologous bone marrow stem cells to the infarcted myocardium followed by directed vasculogenesis. Therefore, it is worth pursuing it in support of the ongoing clinical trials of cardiac regenerative therapy.
ABSTRACT
INTRODUCTION: Embryonal carcinoma of the ovary (ECO), pure or admixed to other tumors, is the deadly gynecological cancer. SPECIFIC AIM: The specific aim of this work was identification, isolation, clonal expansion, and molecular profiling of the pluripotent cells in the embryonal carcinomas of the ovaries. PATIENTS METHODS: The samples were acquired from the patients, who were clinically and histopathologically diagnosed with the advanced, pure embryonal carcinomas of the ovaries. The cell surface display of the TRA-1-60 and SSEA-4 was analyzed by flow cytometry (FCM), immunoblotting (IB), multiphoton fluorescence spectroscopy (MPFS), nuclear magnetic resonance spectroscopy (NMRS), and total reflection x-ray spectroscopy (TRXFS). The transcripts of the Oct4A and Nanog were analyzed by qRTPCR and MPFS and the products by MPFS. The human pluripotent, embryonic stem cells (ESC), human pluripotent, embryonal carcinoma of the testes (ECT), healthy tissues of the ovary (HTO), healthy tissue of testes (HTT), peripheral blood mononuclear cells (PBMC), and bone marrow mononuclear cells (BMMC) served as the controls. RESULTS: The studied embryonal carcinomas of the ovaries (ECOs) contained the cells with the strong surface display of the TRA-1-60 and SSEA-4, which was similar to the pluripotent ESC and ECT. Their morphology was consistent with the histopathological diagnosis. Moreover, these cells showed strong expression of the Oct4A and Nanog, which was similar to the pluripotent ESC and ECT. The ECO cells formed embryoid bodies, which differentiated into ectoderm, mesoderm, and endoderm. These cells were induced to differentiate into muscles, epithelia, and neurons. CONCLUSION: Herein, we revealed presence and identified molecular profiles of the clones of the pluripotent stem cells in the embryonal carcinomas of the ovaries. These results should help us with refining molecular diagnoses of these deadly neoplasms and design biomarker-targeted, patient-centered, personalized therapy.
