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Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells†‡

  1. Ulf Tiemann1,2,
  2. Malte Sgodda1,
  3. Eva Warlich3,4,
  4. Matthias Ballmaier5,
  5. Hans R. Schöler1,2,6,
  6. Axel Schambach3,4,§,
  7. Tobias Cantz1,2,*

Article first published online: 4 MAY 2011

DOI: 10.1002/cyto.a.21072

Issue

Cytometry Part A

Cytometry Part A

Volume 79A, Issue 6, pages 426–435, June 2011

Additional Information

How to Cite

Tiemann, U., Sgodda, M., Warlich, E., Ballmaier, M., Schöler, H. R., Schambach, A. and Cantz, T. (2011), Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells. Cytometry, 79A: 426–435. doi: 10.1002/cyto.a.21072

Author Information

  1. 1

    Junior Research Group Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, 30625 Hannover, Germany

  2. 2

    Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany

  3. 3

    Junior Research Group Hematopoietic Cell Therapy, Cluster of Excellence REBIRTH, Hannover Medical School, 30625 Hannover, Germany

  4. 4

    Department of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany

  5. 5

    Cell Sorting Core Facility, Hannover Medical School, 30625 Hannover, Germany

  6. 6

    Medical Faculty, University of Münster, 48149 Münster, Germany

  1. §

    Contact for plasmid requests

*Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

  1. †

    Author Disclosure Statement: The authors declare that no conflicting financial interests exist.

  2. ‡

    Author contributions: U.T.: vector production, iPSC generation and characterization, data analysis and interpretation, and manuscript writing. M.S.: qRT-PCR analyses. E.W.: generation of vector plasmids. M.B.: flow cytometry setup and cell sorting. H.R.S.: conception and design. A.S.: generation of vector plasmids and design of the study. T.C.: conceptional design of the study, data analysis and interpretation, and manuscript writing. All authors approved the final version of the manuscript.

Publication History

  1. Issue published online: 18 MAY 2011
  2. Article first published online: 4 MAY 2011
  3. Manuscript Accepted: 4 APR 2011
  4. Manuscript Revised: 25 MAR 2011
  5. Manuscript Received: 2 FEB 2011

Funded by

  • German Research Foundation. Grant Number: DFG: EXC 62/1
  • German Ministry for Education and Research. Grant Number: 01GN0812, 01GM0854 and ReGene
  • DAAD. Grant Number: 0315187

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Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_21072_sm_suppinfofig1.tif4246KSupporting Information Figure 1. Supplementary Figure 1. A: Silencing of RF-coupled fluorophore expression occured in most colonies and indicated fully reprogrammed iPSCs. Efficient silencing strongly correlated with morphology indicative of pluripotent cells. B: Some colonies failed to acquire the characteristic hallmarks of ESC-like morphology and at the same time maintained expression of exogenous fluorescent transgenes. Both examples were derivatives of the same subpopulation Oskm. Scale bar, 100 μm.
CYTO_21072_sm_suppinfofig2.tif1326KSupporting Information Figure 2. Direct comparison of Oct4double and equal RF stoichiometries at different total levels by modified transduction. A: Desired MOI ratios for the 4 RFs could be confirmed by flow cytometric analysis measuring the associated fluorophores in equally treated control samples. B: Colony count after transduction with either 1 : 1 : 1 : 1 or Oct4double Sox2single Klf4single c-Mycsingle RF stoichiometries. The throughout higher numbers of AP-positive and ESC like colonies in Oct4double samples were tested for statistical significance by performing a paired, two-tailed Student's t-test.
CYTO_21072_sm_suppinfofig3.tif2002KSupporting Information Figure 3. Modified transduction of human fetal fibroblasts intending distinct RF stoichiometries. Measured MOI ratios of the RFs closely resembled the planned ones. B: Examples of AP-positive colonies obtained from samples transduced with different RF ratios. Scale bar, 100 μm. C: Colony count after AP staining. Excessive Oct4 levels yielded higher colony numbers.
CYTO_21072_sm_suppinfofig4.tif4682KSupporting Information Figure 4. mRNA levels in spontaneously differentiating embryoid bodies generated from iPSCs with distinct RF stoichiometries (day 5, normalized to ESC-derived EBs). Markers for all three germ layers and for the germ-line could be detected. Error bars display standard deviations among triplicate samples. Gata4: GATA-binding protein 4, Afp: a fetoprotein, T: Brachyury, Kdr: VEGF-receptor, Trp63: keratinocyte transcription factor, Otx2: orthodenticle homeobox 2, Dppa3: Developmental pluripotency-associated protein 3 (Stella). B: Directed differentiation of iPSCs into more mature cell types was initiated by embryoid body formation and yielded neurons (C), contracting cardiomyocytes (D), and hepatic progenitors (E). Tubβ3: tubulin β3, cTnT: cardiac troponin T, AFP: a-fetoprotein. The exemplary pictures show an iPSC line established from the sorting fraction OskM. Scale bar, 75 μm.
CYTO_21072_sm_suppinfo.doc37KSupporting Information

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