title: Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression ofDNASE1,DNASE1L3,DNASE2,DFFBControlled ByPOLA1Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinantDNASE1, DNASE1L3, DNASE2, DFFBcontrolled byPOLApromoter; (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, recombinantDNASE1, DNASE1L3, DNASE2, DFFBcontrolled byPOLApromoter 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 byPOLApromoter, 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Phoenix Biomolecular Engineering Foundation, San Francisco, CA 94105, USA ; University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Marek
authors.initials: M
authors.lastname: Malecki
authors.affiliation: University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: LaVanne
authors.affiliation: University of Wisconsin, Madison, WI 53706, USA.
authors.forename: Dominique
authors.initials: D
authors.lastname: Alhambra
authors.affiliation: South Dakota State University, Brookings, SD 57007, USA.
authors.forename: Chaitanya
authors.initials: C
authors.lastname: Dodivenaka
authors.affiliation: South Dakota State University, Brookings, SD 57007, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Nagel
authors.affiliation: Phoenix Biomolecular Engineering Foundation, San Francisco, CA 94105, USA ; San Francisco State University, San Francisco, CA 94123, USA.
authors.forename: Raf
authors.initials: R
authors.lastname: Malecki
keywords: Cancerogenesis; DNA polymerase; DNase; Neoplasm; Pluripotent stem cell; Regenerative medicine; Stage specific embryonal antigen; Stem cell induced cancerogenesis; Stem cell therapy; Tumorigenesis