Entschlüsselung der molekularen Grundlagen der hypertrophen Kardiomyopathie mit Hilfe von induzierten pluripotenten Stammzellen

• Antragsteller: Dr. Sebastian Diecke
• Fachliche Zuordnung: Zellbiologie
• Förderung: Förderung von 2012 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 218479891

• Erstellungsjahr: 2015

Zusammenfassung der Projektergebnisse

In summary, the 4-in-1 CoMiP is an effective alternative reprogramming method for deriving integration-free iPSCs from various donor tissue sources, including PBMCs under chemically defined conditions without using animal-derived products. Compared to other DNA-based reprogramming methods like the minicircle or the Yamanaka three individual plasmids system, the CoMiP construct is faster and less expensive for inducing pluripotency in a somatic cell type. Furthermore, the cost effectiveness of this new reprograming technique should be emphasized because of the current impetus for major research institutions to generate large-scale iPSC banks. For example, the estimated cost for the consumables per derived iPSC line is ~$80 for 4-in-1 CoMiP versus $500 for Sendai virus. Hence this CoMiP construct will allow novice and inexperienced researchers alike to easily obtain bona fide iPSCs within 14 days. Aside from being the fastest reprogramming method, the 4-in-1 CoMiP plasmid does not result in intermediate iPSCs and, due to its color label, the entire reprogramming process is easy to monitor. With all these superior qualities, we believe that this new technology is of special interest given the great potential of iPSCs in regenerative medicine. I used most of my time to develop an integration free reprogramming technique. Moreover I changed the type of disease and the mutation I was working on. As mentioned above the reason for that was that the proposed project was already addressed and nearly published by a different postdoc. Therefore I decided to switched to DCM as a disease and the LMNA mutation because we had access to large family with a novel LMNA mutation causing a severe cardiac phenotype. Unfortunately I was not able to detect any obvious in vitro phenotype by comparing the iPSCs derived cardiomyocytes from either the control or effected patient´s. That’s why I started to generate isogenic cell lines in the assumption to increase any possible phenotype by diminishing the genetic variability between different patient samples. This process was very time consuming and therefore the functional analyses are still under preparation. Nevertheless this project already showed interesting data.

Projektbezogene Publikationen (Auswahl)

• Pushing the reset button: chemical-induced conversion of amniotic fluid stem cells into a pluripotent state. Mol Ther. 2012 Oct;20(10):1839-41
  Diecke S, Wu JC
  
• Costimulation-adhesion blockade is superior to cyclosporine A and prednisone immunosuppressive therapy for preventing rejection of differentiated human embryonic stem cells following transplantation. Stem Cells. 2013 Nov;31(11):2354-63
  Huber BC, Ransohoff JD, Ransohoff KJ, Riegler J, Ebert A, Kodo K, Gong Y, Sanchez-Freire V, Dey D, Kooreman NG, Diecke S, Zhang WY, Odegaard J, Hu S, Gold JD, Robbins RC, Wu JC
  
• The role of SIRT6 protein in aging and reprogramming of human induced pluripotent stem cells. J Biol Chem. 2013 Jun 21;288(25):18439-47
  Sharma A, Diecke S, Zhang WY, Lan F, He C, Mordwinkin NM, Chua KF, Wu JC
  
• Chemically defined generation of human cardiomyocytes. Nat Methods. 2014 Aug;11(8):855-60
  Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, Mordwinkin NM, Plews JR, Abilez OJ, Cui B, Gold JD, Wu JC
  
• Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts. Methods Mol Biol. 2014;1181:1-13
  Diecke S, Lisowski L, Kooreman NG, Wu JC
  
• Transplanted terminally differentiated induced pluripotent stem cells are accepted by immune mechanisms similar to self-tolerance. Nat Commun. 2014 May 30;5:3903
  de Almeida PE, Meyer EH, Kooreman NG, Diecke S, Dey D, Sanchez-Freire V, Hu S, Ebert A, Odegaard J, Mordwinkin NM, Brouwer TP, Lo D, Montoro DT, Longaker MT, Negrin RS, Wu JC
  
• Improved approach for chondrogenic differentiation of human induced pluripotent stem cells. Stem Cell Rev. 2015 Apr;11(2):242-53
  Nejadnik H, Diecke S, Lenkov OD, Chapelin F, Donig J, Tong X, Derugin N, Chan RC, Gaur A, Yang F, Wu JC, Daldrup-Link HE
  
• Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Sci Rep. 2015 Jan 28;5:8081
  Diecke S, Lu J, Lee J, Termglinchan V, Kooreman NG, Burridge PW, Ebert AD, Churko JM, Sharma A, Kay MA, Wu JC.