02 Pluripotent Stemc Cells_211010

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					Induced Pluripotent Stem Cells
         (IPS Cells)


                     Dr. Moritz Frech
                       21.10.2010




   Albrecht-Kossel-Institute for Neuroregeneration (AKos)
                   University of Rostock
Stem Cell Classifications
        Totipotent stem cells: can differentiate into any cell type
        in the body; can grow to complete individuals (in the
        appropriate environment); generally cells from the first few
        divisions after fertilization (maximum 8-cell state in humans)


        Pluripotent stem cells: descendents of totipotent stem cells
        which develop by about Day 4 after fertilization; can
        differentiate into any cell type other than totipotent cells

         Multipotent stem cells: descendents of pluripotent stem
         cells and antecendents of specialized cells in particular
         tissues; Ex: hematopoietic stem cells found in bone
         marrow give rise to all forms of blood cells

         Progenitor cells “unipotent stem cells”: can produce only
         one cell type; Ex: erythroid progenitor cells differentiate
         into only red blood cells


                                                                             2
        University of Rostock                    Albrecht-Kossel-Institute
 Origin of Human Stem/Progenitor Cells

early blastocyte with embryoblast
    and trophoblast (~4 days)




fetal progenitor cells e.g. neural
progenitor cells from midbrain or
       cortex (~ 10 weeks)




                       Ethic issues




                                                                    3
                University of Rostock   Albrecht-Kossel-Institute
If you have 3 wishes ...


     Easy access to pluripotent cells without any ethic issues

              Autologous cells to avoid rejection in host

      Cells with high potential to differentiate in any cell type




               Induced Pluripotent Stem Cells ?




                                                                            4
        University of Rostock                   Albrecht-Kossel-Institute
Can terminated cells be changed?
                       Iwasaki and Akashi, Immunity 2003
       GATA-1 Converts Lymphoid and Myelomonocytic Progenitors into the
                     Megakaryocyte/Erythrocyte Lineages

  Ectopic expression of transcription factors can reprogram cells into another lineage




            General mechanism to reprogramm other cell types?




                                                                                         5
         University of Rostock                          Albrecht-Kossel-Institute
First description of successful reprogramming




                                            Cell 2006

  re-programming of murine fibroblasts in cells with characteristics of embryonic stem cells



                              - Yu and Thomson, Science 2007
                           - Takahashi and Yamanaka, Cell 2007
                                      - Park, Nature 2008
                                   - Lawry and Plath, 2008

       reproduced the iPS phenomenon in human dermal fibroblasts by ectopic
         expression of four embryonic stem cell specific transcription factors.


PubMed search Oct. 2010 ~1900 references for search string: induced pluripotent stem cells
                                     ~ 10/ week


                                                                                               6
            University of Rostock                         Albrecht-Kossel-Institute
How does it work?

 Takahashi and Yamanaka reported that pluripotent stem cells can be established by 4
      transcription factors selected from a list of 24 mouse ES enriched genes:

                Oct4: transcription factor -> maintains pluripotent status
                Sox2: transcription factor -> maintains pluripotent status
                Klf4: transcription factor -> maintains pluripotent status
                        Myc:oncogen -> high reproduction of cells




                                                 verändert nach Zaehres and Schoeler, Cell 2007




                         Mechanisms are yet not fully understood


                                                                                                  7
         University of Rostock                                Albrecht-Kossel-Institute
Comparison of hIPS and hES cells




                                                                   verändert nach Zaehres and Schoeler, Cell 2007

              IPS are similar to hES cells regarding:

                                    Proliferation
                                    Morphology
                                    Pluripotency
                                 Surface markers
                                Expression profile
                                 Epigenetic status
                               Differentiation in vitro
                               Teratoma formation




                                                                                                               8
       University of Rostock                              Albrecht-Kossel-Institute
Technical approach: Transduction with Lent-viral vectors
                                                                     verändert nach Product manual Thermo Fisher Scientific

•   Transduction of target cells by Lenti-viral vectors



              GFP as control of transfection
                      GFP


                                               +


                                                                    human fibroblasts



                                                          human fibroblasts transfected
                                                             with GFP control vector
                                                   -> determination of needed amount of vector



                                                                                                                    9
           University of Rostock                          Albrecht-Kossel-Institute
    Technical approach: Transduction
                                                                                  verändert nach Product manual Thermo Fisher Scientific

•    Transduction with a combination of factors

                                                             - not all factors are needed
     Used by Takahasi et al.


                                                             - combination is different for each target cell

                                                             - combination depends on the                            intrinsic
                                                             expression of transcription factors



      non- transfected                    72h after transfection




                                                                                                                                10
                      University of Rostock                            Albrecht-Kossel-Institute
Technical approach: IPS cell colonies
                                                         verändert nach Product manual Thermo Fisher Scientific

          Growth of colonies of hIPS cells on mice feeder layer




     Growth of colonies of hIPS cells in a feeder free culture system




        Further steps of cultivation are the same as for hES cells

                                                                                                       11
        University of Rostock                 Albrecht-Kossel-Institute
Technical approach: Marker of pluripotency
     hIPS cells should express same pluripotency markers as hES cells

                                  Alkaline
                                phosphatase                          RT-PCR




                                              other pluripotency markers
                                                       Tra-1-81
                                                       Tra-1-60
                                                        SSEA3
                                                        SSEA4
                                                                verändert nach Product manual Thermo Fisher Scientific



                                                                                                              12
        University of Rostock                        Albrecht-Kossel-Institute
Technical approach: Gene expression and epigenetic status
           Molecular analyses and comparison of human induced pluripotent stem
                              cells and embryonic stem cells




verändert nach Chin et al, Cell Stem Cell 2009



                                                                                         13
                        University of Rostock                Albrecht-Kossel-Institute
Technical approach: Capability to form all three germ layers

 Pluripotent cells should be able to generate cells of all three germ layers

 - Generation of chimeric embryos in mice by incorporation of IPS cells in blastocyste

  -> IPS cell contribution to tissues out of three germ layers was confirmed by GFP genotyping




                                                                               verändert nach Zhou et al. 2009


 -Teratoma formation:
 Teratoma is an encapsulated tumor with tissue or organ components resembling
 normal derivatives of all three germ layers.
                                                                  1

                                     1 mesoderm: cartilage
                                     2 endoderm: glands
                                     3 ectoderm: skin                                       3
                                                                           2

                                                                                                                 14
             University of Rostock                           Albrecht-Kossel-Institute
Technical approach: Capability to differentiate
     Pluripotent cells should be able to differentiate into cells of all three germ layers

                                                                  verändert nach Takahashi und Yamanaka et al. 2006




                      mesoderm              endoderm
                                                                    ectoderm



                            Protocols for directed differentiation needed



                                                                                                                      15
           University of Rostock                             Albrecht-Kossel-Institute
Pros and Cons of genetic reprogramming by lentivirus

 - Meanwhile established method

 - “High” efficiency of 0.01%

 - Rapid transduction




 - Transduced factors are tumorigenic or tumor promoting

 - Retroviral vector inserted randomly into the genome may activate
 oncogenes

 - Genetically reprogrammed IPS cells are thus not suitable for
 therapeutical applications



                                                                               16
          University of Rostock                    Albrecht-Kossel-Institute
Alternative ways of reprogramming




                                                                17
       University of Rostock        Albrecht-Kossel-Institute
Recombinant proteins


•   To avoid introducing exogenous genetic modifications,
    reprogramming factors are delivered directly into the target cells.

•   HIV-tat peptide, a basic Arginine rich sequence, has been
    demonstrated to act as entrance sequence for protein transduction.

•   In this aproach a short poly- Arginine (11R) tail is conjugated
    to the C-Terminus of the reprogramming factors
    Oct 3/4, Sox2, Klf4 and c-Myc




                                                                           18
        University of Rostock                  Albrecht-Kossel-Institute
    Recombinant proteins: Technical approach
•    Reprogramming factors have been delivered to Oct4-GFP reporter MEF by
     adding them directly to the media

•    Reprogramming normally requires 7-10 d
     The proteins are added in cycles to provide a sufficient factor level

•    After 30-35 days first GFP+ colonies were observed, picked and further
     cultured in mESC conditions.
                                                 Phase                  GFP
Timeline




                                                                                     19
             University of Rostock                       Albrecht-Kossel-Institute
  Recombinant proteins: Confirmation of pluripotency

Indistinguishable from mESC in terms of :
• Morphology
• Pluripotency marker expression
• Differentiation


                                                  neural progenitor cells mature neurons




                                                   cardiomyocite           Endoderm
                                                         s




                                                   Pancreatic cells         Liver cells

                                    Immunocytochemistry


                                                                                           20
            University of Rostock                             Albrecht-Kossel-Institute
Recombinant proteins: Human IPS Cells




                                                                21
        University of Rostock       Albrecht-Kossel-Institute
Recombinant proteins: Human IPS Cells

Stable HEK293 cells expressing each of the four factors fused
with a 9R sequence and a myc tag

Target cell line Human newborn fibroblasts (HNF)

HNF were treated with HEK293 cell extracts in several cycles




                                                                               22
          University of Rostock                    Albrecht-Kossel-Institute
    Recombinant proteins: Human IPS Cells


•   Cells differentiated in cells of all three germ layers in vitro and in vivo
    (Teratoma formation in SCID mice)




                                                                                  23
              University of Rostock                   Albrecht-Kossel-Institute
Pro and Con of recombinant proteins

• Save method without using genetic modifications
 -> potentially useful for patient applications

•   But…

•   Even more time consuming than „classical“ IPs generation esp.
    in human cells

•   Transduction efficency is even less 0.001% compared to 0.01%
    with virus based method




                                                                        24
         University of Rostock              Albrecht-Kossel-Institute
Alternative ways of reprogramming




Transient transfection of expression plasmids coding for the transcription factors
required to induce pluripotency. This ‘nonintegration method’ utilized an
adenovirusmediated gene delivery system, and the adenoviral vector showed no signs
of integrating into the host




                                                                                     25
          University of Rostock                     Albrecht-Kossel-Institute
Alternative ways of reprogramming




 Transposons, mobile genetic elements that insert themselves into the genome to
 deliver the reprogramming factors. These genomic additions are removed by the
 enzyme transposase once pluripotency has been attained. The piggyBac
 transposon/transposase system has been used to create both mouse and human iPS
 cells that have passed rigorous tests for similarity to ES cells

                                                                                  26
         University of Rostock                     Albrecht-Kossel-Institute
Alternative ways of reprogramming




  Doxycycline-inducible lentiviral vectors to control the expression of reprogramming
  factors and subsequently excises the virus using Cre-recombinase.


                                                                                        27
          University of Rostock                         Albrecht-Kossel-Institute
Breaktrough in reprogramming?




             RNA-induced pluripotentstem cells (RiPSCs)



                                             no insertion in genome


                                                              1-4%




                                                                               28
       University of Rostock                       Albrecht-Kossel-Institute
Use of IPS Cells

   •   in vitro model to study process of differentiation

   •   in vitro model for substance screening

   •   in vitro disease model

   •   Patient derived IPS cells:
       -> never ending source of autologous cells for transplantation




                                                                            29
         University of Rostock                  Albrecht-Kossel-Institute
Patient derived IPS cells




        ! but same hurdles as for human embryonic stem cells !

                                                                       30
        University of Rostock              Albrecht-Kossel-Institute

				
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