Marek Malecki

TRA-1-60+, SSEA-4+, Oct4A+, Nanog+ Clones of Pluripotent Stem Cells in the Embryonal Carcinomas of the Ovaries

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.

Isolation of single, intact chromosomes from single, selected ovarian cancer cells for in situ hybridization and sequencing

The first step towards effective therapy of cancer is to reveal molecular profiles of all cell clones propelling tumor growth. The specific aim of this project was to develop a technology helping us to isolate patients single, living cells based upon their cancer-specific, cell surface biomarkers, to reveal their molecular profiles, and to isolate, from these selected cells, intact chromosomes for in situ hybridization (FISH) and for next generation sequencing (NGS). We attained this aim, while probing the cells from the ovarian cancer patients. Ovarian cancer is the most deadly of all gynecological cancers. In most of the patients with the advanced stages of this cancer, the gene for epidermal growth factors receptor (EGFR) is mutated, as the deletion variant III, resulting in the truncated transcripts and products. From these patients, we collected cells from peritoneal fluid, blood, lymph, and biopsies. We genetically engineered fluorescent and superparamagnetic single chain variable fragments (scFvs) targeting EGFRwt and EGFRvIII. Using these scFvs, we isolated single, living ovarian cancer cells and analyzed their transcripts and products. We further genetically engineered scFv targeting dsDNA. Using these scFvs, we isolated the entire single, intact chromosomes from the selected, single ovarian cancer cells for NGS and for liquid phase FISH. This novel work-flow opens new routes not only for molecular profiling of the entire spectrum of cancer cell clones in the diagnosed patient, one cell clone at a time, but also for manufacturing targeted contrast for in vivo imaging and for designing and guiding targeted delivery of therapeutic genes in cancer therapy.

Directed cardiomyogenesis of autologous human induced pluripotent stem cells recruited to infarcted myocardium with bioengineered antibodies.

ObjectiveMyocardial infarctions constitute a major factor contributing to non-natural mortality world-wide. Clinical trials ofmyocardial 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 tetravalent 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.MethodsPeripheral 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 model 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.ResultsThe 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 the htAbs, than with isotype antibodies or plain buffers. The retained cells differentiated into cardiomyocytes.ConclusionsThe 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.

Recruitment and retention of human autologous CD34+ CD117+ CD133+ bone marrow stem cells to infarcted myocardium followed by directed vasculogenesis: Novel strategy for cardiac regeneration

BackgroundOngoing 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 aimsThe 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 methodsCardiac 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.ResultsAdministration 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.ConclusionsThis novel strategy improved retention of the patients’ autologous bone marrow 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.

Eradication of Human Ovarian Cancer Cells by Transgenic Expression of Recombinant DNASE1 , DNASE1L3 , DNASE2 , and DFFB Controlled by EGFR Promoter: Novel Strategy for Targeted Therapy of Cancer?

Introduction Ovarian cancer is the most deadly among all gynecological cancers. Patients undergoing systemic therapies of advanced ovarian cancers suffer from horrendous side effects. Cancer survivors and their offspring suffer from iatrogenic consequences of systemic therapies: genetic mutations. The ultimate goal of our work is development of therapies, which selectively and completely eliminate cancer cells, but do not harm healthy cells. An important consideration for attaining this goal is the fact that ovarian cancer cells over-express EGFR or its mutants, what becomes the factor discriminating them from healthy cells - a potential facilitator of personalized therapy. Specific aim The specific aim of this project was threefold: (1) to bioengineer suicide genes’ carrying vectors guided by synthetic antibodies for EGFRvIII and EGFR; (2) to genetically engineer DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, and DFFB controlled by the EGFR promoter; (3) to selectively eradicate ovarian cancer cells by intranuclear targeting of the transgenically expressed recombinant DNases. Methods Synthetic antibodies for EGFR and EGFRvIII were selected from the human library and used to bioengineer biotag-guided transgenes’ vectors. Coding sequences for the human DNASE1, DNASE1L3, DNASE2, DFFB controlled by the EGFR promoter were amplified from the human cDNA and genetically engineered into the plasmid constructs also coding for the fusions with NLS and GFP. The vectors carrying transgenes for the DNases were delivered in vitro into human ovarian cancer cells from ascites and cultures. Results Synthetic antibody guided vectors delivered the transgenes for the recombinant DNases efficiently into the ovarian cancer cells. Transgenic expression and nuclear targeting of the DNases in those cells resulted in destruction of their genomes and led to their death, as validated by labeling with the molecular death tags. In healthy cells, which did not over-express EGFR, no changes were recorded. Conclusion Targeted expression of the recombinant DNASE1, DNASE1L3, DNASE2, DFFB in the ovarian cancers in vitro resulted in their complete eradication, but had no effects upon the healthy cells. This novel therapeutic strategy has a potential for streamlining it into in vivo trials, as personalized, targeted therapy of ovarian and other cancers.

Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2,DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death

Introduction The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors. Specific aim The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases). Methods The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers’ bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies’ guided DNA vectors delivered the transgenes for the human recombinant DNases’ into proliferating stem cells. Results Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells’ genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die. Conclusion Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.