Embryonic & and induced pluripotent Stem Cells. May 2010 Dipl. Biol. Dr. Kurt Pfannkuche

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1 Embryonic & and induced pluripotent Stem Cells May 2010 Dipl. Biol. Dr. Kurt Pfannkuche

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3 What makes a stem cell? open chromatin structure plasticity! Seite 3 self-renewal

4 Plasticity Seite 4

5 Culture of pluripotent cells Murine ES cells - Feeder? - LIF to stimulate STAT pathway - Serum or Serum-free conditions Human ES cells - Feeder? - No LIF - Serum free conditions (Transplantation grade cells?) Seite 5

6 Genetical engineering of ES cells Seite 6

7 Genetical engineering of ES cells Seite 7

8 Spontaneous differentiation of ES cells sets on in embryoid bodies. Undifferentiated murine cells Embryoid bodies (EBs) Seite 8

9 Differentiation in embryoid bodies Seite 9

10 Quality Assay of ES cell cultures Theoretically ES cells can be maintained in culture for a unlimited number of passages Culture of ES cells leads to adaptation Surveillance of the karyotype is mandatory especially in human ES cultures Cryopreservation of human ES cells is one bottleneck of cell quality Seite 10

11 Example 1: Single hes Seite 11 Why they die? No clonal growth of human ES!

12 Example 2: Clumps Seite 12 Why they die? Freezing of clumps not optimal.

13 Example 3: Apoptosis inhibition Seite 13

14 Aberations in chromosome number Murine ES +1 Chromosome Murine ES tetraploid chromosome set Seite 14

15 Fluorescent hybridisation analysis: human ES - normal Seite 15

16 Fluorescent hybridisation analysis: human ES - abnormal Seite 16

17 EBs develop a variety of cell types Cardiac cells in suspension EBs Endothelial cells in plated EBs Smooth muscle cells from EBs All pictures taken by Kurt Pfannkuche. Endothelial cells provided by Manoj Gupta, neuronal cells provided by Aret Beckcioglu, smooth muscle cells provided by Shiva Potta. Cardiac cells in plated EBs Neuronal cells (red) in plated EBs Seite 17

18 Investigation of murine ips cardiomyocytes Seite 18

19 Lineage selection of ES derived cardiomyocytes Seite 19

20 Lineage selection of ES derived cardiomyocytes Seite 20 Foto: Dr. Eugen Kolossov, Axiogenesis AG, Germany

21 Cell May 14;141(4): Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Oshima K, Shin K, Diensthuber M, Peng AW, Ricci AJ, Heller S. Stanford University School of Medicine, Stanford, CA 94305, USA. Seite 21

22 Induction of pluripotency in differentiated cells Seite 22 from J. B. Gurdon et al., Science 322, (2008)

23 Somatic cell nuclear transfer (SCNT) Seite 23 Jianyuan Li, Xuexia Liu, Haiyan Wang, Shouxin Zhang, Fujun Liu, Xuebo Wang, Yanwei Wang. Cloning and Stem Cells. March 2009, 11(1)

24 Somatic cell nuclear transfer: Applications CRYOZOOTECH is happy to announce the birth of the clone of 2003 dressage world champion POETIN 2, a Brandeburg mare that died prematurely in Now CRYOZOOTECH enters the dressage world. This achievement illustrates the use of the cloning technique for genetic. Seite 24

25 Problems associated with SCNT Cloning efficiency is extremly low Oocytes are difficult to derive in larger quantities Oocyte quality (fresh!) is limiting success In human: Generation of human embryos raises severe ethical concerns No ES cells have been derived and characterized from human SCNT blatocysts SCNT approaches may be usefull to understand the process of reprogramming by oocyte plasma Seite 25

26 Induced pluripotency by defined transcription factors Cultured primary cells like skin fibroblasts Seite 26

27 Induced pluripotency by defined transcription factors ips from adult murine fibroblasts Seite 27

28 Induction of pluripotency by viral transduction Lentiviral transduction Highly efficient Good transduction efficiencies even with hard to transfect cells Integration into genome and potential risk of aberant expression of endogenous genes! Incomplete silencing and reactivation risk. Seite 28

29 Induction of pluripotency by viral transduction Adenoviral transduction No integration into genome Very low risk of aberant expression Extremely low efficient Effective to reprogram embryonic but not adult cells Seite 29

30 Induction of pluripotency by transposable elements + transposase enzyme Seite 30

31 Combination of piggy Bac transposon with tetracyclin responsible promoter K Woltjen et al. Nature 000, 1-5 (2009) doi: /nature07863 Seite 31

32 Removal of transposable element after successfull reprogramming Seite 32 From Woltjen et al. Nature 2009

33 Isolation of keratinocytes from plucked human hair Plucked human hair Arrow: Outer root sheath. From the root sheath keratinocytes build the keratine of the hair. Seite 33 Keratinocytes from plucked hair From: Aasen T, Belmonte JC. Nat Protoc. 2010;5(2):

34 Generation of ips cells from hair keratinocytes a) keratinocytes b) keratinocytes infected with GFP-expressing lentivirus c) reprogrammed colonies d) reprogrammed colony e) alkaline phosphatase f) embryoid body formation Seite 34 From: Aasen T, Belmonte JC. Nat Protoc. 2010;5(2):

35 Seite 35 Thank you for your attention!