Potential applications of   germline-cell derived pluripotent stem cells inpreclinical animal models          Fiorella Alt...
Pluripotent stem cells      •Embryonic stem (ES) cells      •Embryonic germ cells derived from primordial germ cells      ...
Spermatogonial stem cells convert spontaneously to GPSCs in long term culture                                             ...
Characteristics of GPSCs        •Express Oct4, Nanog,SSEA-1, Lin28, c-myc        •Positive for Alkaline phosphatase       ...
…GPSCs show high plasticitySarcomeric                                           cardiac troponin T α-actininpan-cadherin  ...
GPSC-derived-NeuronsAstrocytesOligodendrocytesStreckfuss-Bomeke et al.,Stem Cell Res 2009
Do GPSCs generate functional      hepatocytes?
Hepatocyte differentiation protocol                      GPSCStatic                           2 dayssuspensionculture     ...
Morphological and molecular characterisation of GPSCs       during induced hepatocyte differentiation        A            ...
Percentage of hepatocytes in the embryoid bodies                                          GFP+ hepatocyte-                ...
Functional Analyses                                                                                                 Haptog...
How far are we from in vivo-  derived hepatocytes?
12    Non-liver-like     response3456      Liver-like7     response      ES-specific8       liver-like       response
Genes upregulated in GPSC-derived hepatocytes and in primary hepatocytes                     GPSC-
GPSC-                                        Pearson with E16 Hepatocytes                                        0.74     ...
ConclusionsGPSCs can be induced to differentiate intofunctional hepatocytes in vitro :• they express liver specific genes•...
Ongoing projects
Cell therapy and correction of genetic liver diseases     Mouse models : Glycogen storage disease type 1a (G-6-Pase KO    ...
Glycogen Storage Disease Type 1a            (GSD-1a)• Autosomal recessive disorder• Caused by mutations in the catalytic s...
Perl’s Staining 5 days post injection                                      1                             2                ...
FISH: Y chromosomeMale liver                 Female liver         GPSC-treated female liver
Do GPSCs differentiate into functional renal cells?•Optimise existing protocols•Determine which renal cell types formed•Fu...
Find novel genes involved in       pluripotency maintenance• Genes• miRNAsPluripotency assays                      Collabo...
AcknowledgementsMBC                             COLLABORATORS                       BIDMC, Boston         Anatomia Patolog...
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Altruda fiorella torino gennaio 2011-14° convegno patologia immune e malattie orfane [modalità compatibi

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Altruda fiorella torino gennaio 2011-14° convegno patologia immune e malattie orfane [modalità compatibi

  1. 1. Potential applications of germline-cell derived pluripotent stem cells inpreclinical animal models Fiorella Altruda Molecular Biotechnology Center, University of Turin 22 January 2011
  2. 2. Pluripotent stem cells •Embryonic stem (ES) cells •Embryonic germ cells derived from primordial germ cells •Induced pluripotent stem cells (iPS)•Germline cell-derived pluripotent stem cells (GPSCs) Share features of:Embryonic stem cells Adult stem cells
  3. 3. Spermatogonial stem cells convert spontaneously to GPSCs in long term culture * ES-like colony Oct4 SSC to ES-like transition Nanog SSEA-1 Sox2 Lin28 C-myc Oct4 Sox2 SSC= Spermatogonial stem cells Lin28 (Unipotent) C-myc GPSC= Germline-derived pluripotent SSC colony stem cells (Pluripotent)
  4. 4. Characteristics of GPSCs •Express Oct4, Nanog,SSEA-1, Lin28, c-myc •Positive for Alkaline phosphatase •Teratoma formation in vivo •Chimera formation Advantages of GPSCs versus ES cells and other adult stem cells• GPSCs are already pluripotent (no “reprogramming” required)• GPSCs can be passaged for over 30 times without loss in replicative capacity or change in karyotype• GPSCs can be frozen and thawed• GPSCs are derived from adult tissues: Avoid ethical concerns over embryo use for production of pluripotent stem cells• And...
  5. 5. …GPSCs show high plasticitySarcomeric cardiac troponin T α-actininpan-cadherin Cx43 Immunostaining of cardiac clusters Guan et al, Circ. Res 2007
  6. 6. GPSC-derived-NeuronsAstrocytesOligodendrocytesStreckfuss-Bomeke et al.,Stem Cell Res 2009
  7. 7. Do GPSCs generate functional hepatocytes?
  8. 8. Hepatocyte differentiation protocol GPSCStatic 2 dayssuspensionculture Embryoid bodies 4 daysPlated a-FGF + b-FGF (early)on gelatinor GF reduced- 4 daysmatrigel HGF (intermediate) 6 days OSM/Dex/ITS (late)Culture inCollagen gel Modified from Chinzei et al. Hepatology 2002
  9. 9. Morphological and molecular characterisation of GPSCs during induced hepatocyte differentiation A GPSC-derived hepatocytes GPSC NT Day 7(gelatin) Day 21(gelatin) Day 21(collagen) B Days of differentiation GPSCNT 2 7 14 21 28 Liver ALB AFP ALB: Albumin TAT AFP: Alpha fetoprotein Hx TAT: Tyrosine aminotransferase Hx: Hemopexin Hp Hp: Haptoglobin Oct4 Actin Cyp7a1 C Relative Quantity 10000 1000Liver-specific 100 10 1 Cyp7a1: cytochrome P450, family 7, subfamily A, polypeptide 1 0,1 NT 28 NT 28 liver GPSC#1 GPSC#2 Fagoonee et al. Stem Cells and Days of differentiation Development 2010
  10. 10. Percentage of hepatocytes in the embryoid bodies GFP+ hepatocyte- EB 21d like cell neuron-like cell Lentivirus: pCCL.ET.GFP.Sin Enhanced transthyretin promoter Kind gift from L.Naldini 90 Undifferentiated GPSCs hepatocytes (%) 80 Control Infected GFP-positive positive 70 60 0.04% 7.77% 50 FL2-H FL2-H 40 30 20 10 R4 R4 0 NT 21 35 100 101 102 103 104 100 101 102 103 104 Days of differentiation FL1-H FL1-H GPSC-derived hepatocytes Control Day 21(infected) Day 35(infected) 0.03% 70.16% 82.68% FL2-H FL2-H FL2-H R3 R3 R3 0 1 2 3 4 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 FL1-H FL1-H FL1-H
  11. 11. Functional Analyses Haptoglobin secretion collagen matrigel Albumin secretion gelatin serum Matrigel Collagen gel HpAlbumin (ng)/ mg protein 1000 Urea Synthesis 100 1000 Urea (µg mg protein **a * ***b 10 100 * * µg)/ *** 1 7 11 14 18 24 27 PH 10 Days of differentiation 1 11 14 18 22 PH Days of differentiation Glycogen deposits and Indocyanine green uptake PAS-staining Indocyanine green GPSC-derived hepatocytes MEFs GPSC-derived hepatocytes *
  12. 12. How far are we from in vivo- derived hepatocytes?
  13. 13. 12 Non-liver-like response3456 Liver-like7 response ES-specific8 liver-like response
  14. 14. Genes upregulated in GPSC-derived hepatocytes and in primary hepatocytes GPSC-
  15. 15. GPSC- Pearson with E16 Hepatocytes 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84 ES nt ES EB day 2 ES EB day 7 ES EB day 14 ES EB day 21 ES EB day 28 GPSC1 nt GPSC1 EB day 2 GPSC1 EB day 7 GPSC1 EB day 14 GPSC1 EB day 21 GPSC1 EB day 28 GPSC2 nt GPSC2 EB day 2 than adult primary hepatocytes GPSC2 EB day 7 GPSC2 EB day 14 GPSC2 EB day 21 GPSC2 EB day 28 GPSC-derived hepatocytes are closer to E16 (fetal) primary hepatocytesPearson correlation
  16. 16. ConclusionsGPSCs can be induced to differentiate intofunctional hepatocytes in vitro :• they express liver specific genes• they show functional properties of hepatocytes• they show high efficiency (80%) of differentiation• they are closer to fetal hepatocytesFagoonee et al., Stem Cells and Development 2010
  17. 17. Ongoing projects
  18. 18. Cell therapy and correction of genetic liver diseases Mouse models : Glycogen storage disease type 1a (G-6-Pase KO mice from S.Eva (Gaslini hospital, Genova)) Hemochromatosis (Hfe-null mice from E.Tolosano) Fulminant hepatic failure (galactosamine/LPS) Ongoing Experimental set-up set- Hepatocytic differentiation of GPSCs Intrasplenic or Intraparenchymal injection Partially hepatectomised, monocrotaline-treated female miceDetermine presence of Y chromosome (FISH) in female livers (collaborator: G. Inghirami) Isolation of human SSC and derivation of GPSCs
  19. 19. Glycogen Storage Disease Type 1a (GSD-1a)• Autosomal recessive disorder• Caused by mutations in the catalytic subunit of the glucose-6-phosphatase• glucose-6-phosphatase is a key enzyme in the glucose metabolism• glucose-6-phosphatase is expressed primarily in the liver and kidney• GSD-1°patients manifest hypoglycemia, growth retardation, hepatomegaly and other metabolic disorders• No complete cure have been reported (liver transplantation)
  20. 20. Perl’s Staining 5 days post injection 1 2 Hfe-null mice 3 41-2 negative control (mice treated with monocrotaline, hepatectomised)3-4 positive (mice treated with monocrotaline, hepatectomised and GPSC injection)
  21. 21. FISH: Y chromosomeMale liver Female liver GPSC-treated female liver
  22. 22. Do GPSCs differentiate into functional renal cells?•Optimise existing protocols•Determine which renal cell types formed•Functional analyses•Animal models: renal ischemia and reperfusion injury glycerol-induced acute renal injury Autosomal Recessive Polycystic Kidney Disease Collaborators: G. Camussi S. Bruno
  23. 23. Find novel genes involved in pluripotency maintenance• Genes• miRNAsPluripotency assays Collaborators: Ferdinando Di Cunto Paolo Provero Rosario Piro
  24. 24. AcknowledgementsMBC COLLABORATORS BIDMC, Boston Anatomia Patologica, TurinSharmila Fagoonee Giorgio Inghirami Pier Paolo Pandolfi Robin Hobbs Cristina AbeleLetizia De ChiaraFrancesca Barbieri MBC San Raffaele, MilanMarco Zuin Giovanni Camussi Luigi Naldini Alessio Cantore Stefania BrunoEmanuela TolosanoLorenzo Silengo Paolo Provero IRCC, Candiolo Enzo Medico Ferdinando Di Cunto Daniela Cantarella Rosario Piro Gaslini, Genova Sandra Eva

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