title: Relevance of HCN2-expressing human mesenchymal stem cells for the generation of biological pacemakers.
abstract: BACKGROUND
  The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs.
  
  
  METHODS
  hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone.
  
  
  RESULTS
  Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores.
  
  
  CONCLUSIONS
  mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.
authors.affiliation: Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.
authors.forename: Ieva
authors.initials: I
authors.lastname: Bruzauskaite
authors.affiliation: Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania. daibironai@gmail.com.|Department of Pathology, Forensic Medicine and Pharmacology, Vilnius University, Faculty of Medicine, Vilnius, Lithuania. daibironai@gmail.com.
authors.forename: Daiva
authors.initials: D
authors.lastname: Bironaite
authors.affiliation: Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.
authors.forename: Edvardas
authors.initials: E
authors.lastname: Bagdonas
authors.affiliation: Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Vytenis Arvydas
authors.initials: VA
authors.lastname: Skeberdis
authors.affiliation: Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Denkovskij
authors.affiliation: Institute of Materials Science, Kaunas University of Technology, Kaunas, Lithuania.
authors.forename: Tomas
authors.initials: T
authors.lastname: Tamulevicius
authors.affiliation: Clinic of Rheumatology, Orthopedic and Traumatology and Reconstructive Surgery, Faculty of Medicine, Vilnius University, Vilnius, Lithuania.
authors.forename: Valentinas
authors.initials: V
authors.lastname: Uvarovas
authors.affiliation: Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania.
authors.forename: Eiva
authors.initials: E
authors.lastname: Bernotiene
mesh_terms: D000818:Animals; D001683:Biological Clocks / Q000235:genetics*; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002448:Cell Adhesion; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D018274:Electroporation; D016193:G1 Phase / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D019076:Transgenes
chemical_list: D018797:Cell Cycle Proteins; C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015221:Potassium Channels; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Biological pacemaker; HCN2; Scaffold; Stem cells; Tissue regeneration