CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES

1. CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES L. Gianaroli, MC Magli, AP Ferraretti S.I.S.ME.R. Reproductive Medicine Unit - Via Mazzini, 12 - 40138 Bologna sismer@sismer.it

2. The initial goal was treatment of infertility. However, assisted reproduction techniques are no longer used only to help infertile couples, but they have a great significance in the field of therapeutic medicine. S.I.S.ME.R. VISION 2000 Since the birth of the first baby conceived using IVF techniques in 1978 over three million babies have been born worldwide as the result of ART.

3. Therapeutic medicine S.I.S.ME.R. VISION 2000 - Gamete and tissue cryo-banking - PGD – HLA matching - Stem cells ART

4. Therapeutic medicine S.I.S.ME.R. VISION 2000 - Gamete and tissue cryo-banking ART

5. OOCYTE CRYO-BANKING FERTILITY RESTAURATION S.I.S.ME.R. VISION 2000 Premature ovarian failure - POF defined as menopause before the age of 40 years or hypergonadotropic hypogonadism defined as menopause consequent to chemotherapy Depending on the extent of damage to the ovaries Acute Ovarian Failure (AOF)= loss of ovarian function during or shortly after the end of chemotherapy Premature menopause= loss of ovarian function that occurs years after the end of chemotherapy (before age 40 yr)

6. OOCYTE CRYO-BANKING RISK OF POF ACCORDING TO AGE AFTER CHEMOTHERAPY S.I.S.ME.R. VISION 2000 • The chemotherapeutic destruction of an already low follicular reserve, reduces the number of follicles below a certain ‘threshold’ number required to sustain ovarian function, resulting in menopause. Mattle et al, 2005

7. OOCYTE CRYO-BANKING RISK OF POF ACCORDING TO CHEMOTHERAPEUTIC AGENTS S.I.S.ME.R. VISION 2000 Alkylating agents are extremely gonadotoxic because they are not cell cycle-specific and can damage resting primordial follicles. Cycle-specific agents such as MTX and 5-FU do not have any effect on ovarian reserve. Sonmezer, M. et al. Oncologist 2006;11:422-434

8. OOCYTE CRYO-BANKING DOSE-EFFECT DAMAGE ON OVARIAN FUNCTION S.I.S.ME.R. VISION 2000 No. of primordial follicles Dose of Cyclophosphamide (mg/kg) Follicular damage from alkylating agents is dose-dependent. A dose of chemotherapy strong enough to destroy 50% of the ovarian primordial reserve does not affect the reproductive performance in a murine model. Meirow et al, 1999

9. OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING S.I.S.ME.R. VISION 2000 • Ovary(s) removed laparoscopically, divided into small strips, frozen and stored • Females, before and after puberty • Outpatient surgical procedure • Experimental, one possible live birth to date • Re-implantation can restore hormone function

10. OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING S.I.S.ME.R. VISION 2000

11. OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING – CLINICAL RESULTS S.I.S.ME.R. VISION 2000 • A transient restoration of spontaneous ovarian follicular development and estrogen production, but not ovulation, was observed after autotransplantation of frozen/thawed ovarian tissue that had been harvestedand banked before chemotherapy and radiation therapy for lymphoma (Redford et al., 2001). • Embryo development was obtained after autotransplantation of frozen/thawed ovarian tissue (Oktay and Sommezer, 2004).

12. OOCYTE CRYO-BANKING OVARIAN TISSUE FREEZING – CLINICAL RESULTS S.I.S.ME.R. VISION 2000 • Livebirth resulted after orthotopic transplantation of cryopreserved ovarian tissue (Donnez, 2004). • A pregnancy was obtained after transplantation of cryopreserved ovarian tissue and IVF in a patient with ovarian failure after chemotherapy (Meirow, 2005). • Actually, follicular growth, hormonal production and some pregnancies (5 as reviewed by Fabbri et al., 2008), spontaneous or after IVF treatments, have only been achieved after the autotransplantation technique.

13. OOCYTE CRYO-BANKING OOCYTE FREEZING – CLINICAL APPLICATIONS S.I.S.ME.R. VISION 2000 Oocyte cryopreservation could be a clinical tool for: • Women at risk of losing ovarian function • Women desiring fertility preservation (e.g. delayed maternity) • Eliminating ethical concerns of embryo cryopreservation • Solving the dilemma of abandoned frozen embryos in the IVF laboratory

14. OOCYTE CRYO-BANKING OOCYTE FREEZING – SISMER EXPERIENCE S.I.S.ME.R. VISION 2000

15. S.I.S.ME.R. VISION 2000 OOCYTE CRYO-BANKING OOCYTE FREEZING – VITRIFICATION SISMER EXPERIENCE (2004-2008) % Implantation rate (FHB) Spontaneous abortion rate Clinical pregnancy rate

16. Therapeutic medicine S.I.S.ME.R. VISION 2000 - Gamete and tissue cryo-banking - PGD – HLA matching ART

17. PGD S.I.S.ME.R. VISION 2000 GENETIC INVESTIGATION Diagnosis implies looking for: specific disease mutation (including X-linked diseases) chromosome aberration Screening implies looking for a genetic defect in all members of a population at risk being the risk dependent on the incidence and severity of the defect

18. PGD  High risk of genetic disease Single gene disorders Chromosomal abnormalities Late onset diseases (i.e. Huntington Chorea) • Predisposition to late onset diseases (i.e. cancer)  HLA typing  Aneuploidy • Sex selection for family balancing S.I.S.ME.R. VISION 2000 MAIN INDICATIONS ESHRE PGD consortium data collection 1997-2005 14.419 cycles Goossens et al. (2008) ESHRE PGD Consortium data collection VIII: cycles from January to December 2005 with pregnancy follow-up to October 2006. Hum Reprod 23,2629-2645.

19. PGD WHY TO GO FOR IT? S.I.S.ME.R. VISION 2000 Normally fertile couples whose children might inherit - a severe disease - a predisposition to a pathology Normally fertile couples who wish to save a sibling’s life (HLA-typing)

20. PGD WHY TO GO FOR IT? S.I.S.ME.R. VISION 2000 “IVF aims at having a child, PGD aims at having a healthy child and PGD/HLA testing aims at having a healthy and helpful child”. UNESCO’s report on preimplantation genetic diagnosis (PGD) and Germ-Line Intervention, 2003.

21. PGD FOR HLA - MATCHING S.I.S.ME.R. VISION 2000 • Healthy embryos are selected for transfer avoids the need for termination of an ongoing pregnancy in cases of an affected fetus • HLA – matching with an affected child

22. S.I.S.ME.R. VISION 2000 PREIMPLANTATION HLA MATCHING • One of the most recent applications in reproductive medicine. • Viable option for couples with children needing haematopoietic stem cell (HSC) transplantation. • Selection of embryos both free of disease and HLA matched with the existing child. • PGD is used not only to avoid the birth of affected children, but also to conceive healthy children who may also be potential HLA-identical donors of HSC • At delivery of the newborn, cord blood HSC can be used to treat the affected sibling.

23. S.I.S.ME.R. VISION 2000 Allogeneic HSC transplantation • Only curative option for severe cases of haematopoietic disorders. • A critical factor associated with a favourable outcome is the use of HLA identical donors • HSC from HLA identical siblings provide the higher success rate (~90%) • Reduced incidence of graft rejection and other serious complications associated with transplantation. • Transplantation using non HLA-identical donors is associated with higher morbidity and poorer survival. • Limited availability of HLA-matched unrelated donor, identified from national or international registers.

24. S.I.S.ME.R. VISION 2000 Indication for preimplantation HLA matching • Severe cases of haematopoietic disorders requiring a HLA compatible HSC donor. • Thalassemia • Fanconi anaemia • Wiskott-Aldrich syndrome • Diamond-Blackfand Anemia • X-linked Hyper IgM Syndrome • X-linked adrenoleukodystrophy • X-linked Hypohidrotic Ectodermal Dysplasia with immune deficiency • Aplastic anemia • For diseases such as Acute Lymphoid Leukemia, in which HLA matching becomes the primary indication.

25. S.I.S.ME.R. VISION 2000 Patient’s acceptance policy for HLA program • HSC transplantation is the best treatment option for the affected child (advise transplantation hematologist is required); • HSC transplantation is not urgent; • The family cares unconditionally about all the children.

26. DP DM LMP/TAP DQ DR B C A 1 3 4 5        MHC Class II MHC Class I Class III 3 extra DR genes in some individuals can allow 3 extra HLA-DR molecules S.I.S.ME.R. VISION 2000 Simplified map of the HLA region CLASS I: 3 types HLA-A, HLA-B, HLA-C. CLASS II: 3 types HLA-DP,HLA-DQ,HLA-DR. (also MHC class II genes for HLA-DM, and TAP, and LMP)

27. Polymorphism in the MHC 381 317 Class II Class I No of polymorphisms 185 89 91 45 19 20 2 a b a b a b A B C DR DP DQ S.I.S.ME.R. VISION 2000 The HLA Complex (Human Leukocyte Antigen) - located on chromosome 6 - represents one of the most polymorphic regions of human genome. Comparative DNA sequence analysis of HLA complex has shown the presence of a high number of alleles in this region. In the human population, over 1,200 HLA alleles have been identified 492 alleles 657 alleles

28. DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9 DP DP DP DQ DQ DQ DR DR DR B B B C C C A A A Parents DP-1,8 DQ-3,6 DR-5,4 B-7,2 C-9,8 A-11,10 DP-1,2 DQ-3,4 DR-5,6 B-7,8 C-9,10 A-11,12 DP DP DP DQ DQ DQ DR DR DR B B B C C C A A A DP-2,8 DQ-4,6 DR-6,4 B-8,2 C-10,8 A-12,10 X Children DP DP DP DQ DQ DQ DR DR DR B B B C C C A A A DP-9,8 DQ-7,6 DR-5,4 B-3,2 C-1,8 A-9,10 DP DP DP DQ DQ DQ DR DR DR B B B C C C A A A DP-2,9 DQ-4,7 DR-6,5 B-8,3 C-10,10 A-12,9 S.I.S.ME.R. VISION 2000 Inheritance of MHC haplotypes Genes in the MHC are tightly LINKED and usually inherited in a group. The combination of alleles on a chromosome is an MHC HAPLOTYPE all persons: have 2 haplotypes (1 maternal, 1 paternal in origin)

29. HLA - C HLA - A IVS-I-110 G/A HLA - B HLA - C HLA - A IVS-I-110 G/A HLA - B HLA - C HLA - A IVS-I-110 G/A HLA - B S.I.S.ME.R. VISION 2000 Minisequencing-based Preimplantation HLA matching on single blastomeres Father Mother Child

30. HLA - C HLA - A IVS-I-110 G/A HLA - B HLA - C HLA - A IVS-I-110 G/A HLA - B Child Blast. 3 Heterozygote HLA Identical HLA - C HLA - A IVS-I-110 G/A HLA - B HLA - C HLA - A IVS-I-110 G/A HLA - B Blast. 4 Blast. 8 Normal Homozygote HLA Non Identical Minisequencing-based Preimplantation HLA matching on single blastomeres

32. Reproductive Genetics Institute rgi@flash.net Overall Results and Outcome of Preimplantation Diagnosis for Single Gene Disorders & Preimplantation HLA testing RGI Experience 04/14/2008

33. Therapeutic medicine S.I.S.ME.R. VISION 2000 - Gamete and tissue cryo-banking - PGD – HLA matching - Stem cells ART

34. S.I.S.ME.R. VISION 2000 Stem Cells • Stem Cells • - clonogenic, self-renewing • progenitor cells that generate one • or more specialised cell types • Embryonic stem cells and germline stem cells • - pluripotential and immortal • Organ or tissue specific stem cells • - multipotential

35. S.I.S.ME.R. VISION 2000 Differentiation of Stem Cells Totipotent stem cell Blastocyst Pluripotent stem cell Culture Embryo Embryonic stem cell (ES cell) Embryo (gonads) Pluripotent somatic stem cell CNS PNS Hema. Liver Skin Mesen. etc Primitive germ cell (germline stem cell) Multipotent stem cells

36. S.I.S.ME.R. VISION 2000 SOURCES OF STEM CELLS NERVOUS, HAEMATOPOIETIC, EPITHELIAL, INTESTINAL ADULT TISSUE - SPECIFIC CELL TYPE - TRANSDIFFERENTIATION UMBILICAL CORD HIGH PROLIFERATION VERY HIGH DIFERENTIATON POTENTIAL EMBRYONIC STEM CELLS (ES) INNER CELL MASS

37. S.I.S.ME.R. VISION 2000 STEM CELLS THERAPEUTIC APPLICATIONS CRUCIAL FCTORS NUMBER OF CELLS MEDIUM UMBILICAL CORD LOW ES TISSUES HIGH HIGH LOW MEDIUM PROLIFERATION POTENTIAL

38. S.I.S.ME.R. VISION 2000 Applications for Embryonic Stem Cells and their Derivatives • ES cells for research and discovery • Progenitor cells for drug screening • Progenitor cells for toxicology • Gene products (proteins), growth and • differentiating factors, cell surface • molecules for pharmaceutical use in • regenerative medicine

39. S.I.S.ME.R. VISION 2000 Embryonic Stem Cell Derivatives Applications for Cell and Tissue Therapy • Vehicles for the delivery of gene therapies • - correcting genetic disease • - new immunization strategies • - targeting cancers

40. S.I.S.ME.R. VISION 2000 Making ES Cells and their Derivatives Compatible for Transplantation • Make ES cells from all the necessary HLA subtypes – • subject to advice from transplant divisions about the • number necessary • Remove the cell surface expression of the major • histocompatibility antigens from ES cells and their • derivatives • Make customized ES cells for each and every patient – • therapeutic cloning

41. S.I.S.ME.R. VISION 2000 Embryonic Stem Cells Formed by Nuclear Transfer of Adult Cells (Therapeutic Cloning) • ES cells contribute to all tissues in an apparently normal way • - in chimeras • in vitro • in teratomas • No indication of defects seen in cloned foetuses/offspring • helper cell effects • transdifferentiation phenomenon • May be an extremely efficient way to produce transplantation • compatible cells and tissues for patients

42. S.I.S.ME.R. VISION 2000 Therapeutic Cloning ES cells Compatible transplants Nuclear transfer Source of eggs: self, mother, relative, egg bank Cells eg skin Problems: Inefficient - may need large numbers (50 to several hundreds) of eggs Technically demanding - need to be available in many or all hospitals

43. S.I.S.ME.R. VISION 2000 Patient Specific Stem Cells Disease phenotype Nuclear transfer Screening Patients with diseases of undetermined cause, eg. Glioblastoma Alzheimer’s Disease Parkinson’s Disease Motor Neurone Disease Drug Why How Blastocyst Unaffected neurones

44. S.I.S.ME.R. VISION 2000 Cure of Genetic Diseases Using Stem Cells Insert HoxB4 Homeobox gene DNA repair Differentiate into functional hematopoietic lineage Mouse in a Bubble Inject stem cells to correct Rag2 disease Genetic Correction Cultured skin cells Rag2 Mouse (severe combined immuno deficiency) ES Cells Skin cell or Cell fusion Nuclear transfer (Rideout et al. Cell 2002)

45. Allogenic Cell Therapy S.I.S.ME.R. VISION 2000 Non-transferable embryos in frozen storage Induce tolerance Immune suppression > 200,000 HLA types Brain transplantation Neuronal precursors TH+ dopaminergic neurone MHC gene knockouts Propogate cells to master ES cell bank Select null HLA expression Master bank of HLA typed ES cells Direct Neural stem cells differentiation

46. S.I.S.ME.R. VISION 2000 Transplantation of Cells Derived from Human Embryonic Stem Cells • Induction of tolerance across MHC histocompatibility barriers by pretransfer of ES cell- like cells/haematopoietic cells. • An epithelial progenitor stem cells population (positive for glycoprotein MTS24) has been identified that is competent and sufficient to fully reconstitute the thymus. (Gill et al Nature Immunology June 17 2002).

47. S.I.S.ME.R. VISION 2000 Delivering stem cell therapy: Avoiding immune rejection by reconstructing the immune system The thymus is the site where immune system cells learn the difference between self and non-self Richard Boyd and colleagues have shown that rare progenitor cells can rebuild a thymus

48. S.I.S.ME.R. VISION 2000 LONG-TERM ALLOGENEIC GRAFT ACCEPTANCE USING EMBRYONIC STEM CELL-LIKE CELLS (Fandrich et al. Nature Medicine 8: 171 2002)

49. S.I.S.ME.R. VISION 2000 Trophectoderm biopsy for developmental competence Biopsy for genetic diagnosis Transfer to patient Blastocyst 8-cell 4-cell Inner cell mass Embryos 2-cell Transfer to patient ICSI Use in regenerative medicine (eg. diabetes, Parkinson’s Disease, stroke, respiratory disease, cardiac damage, quadriplegia) IVF Female infertility Idiopathic infertility few eggs Many eggs Immature eggs recovered in natural cycle (no fertility drugs) Fertility drugs given to women to make many mature eggs Reproductive Medicine  Therapeutic Medicine

50. EU hESC line Registry (hESCReg) Primary objective: provide information about all human ESC lines derived and used in Europe which are available to the scientific community. Specific Support Action funded by VI FP European Commission (1.048.000 €, 2007 - 2010) S.I.S.ME.R. VISION 2000 Coordinated by: Joeri Borstlap (BCRT - Technical Coord.) Anna Veiga (CRMB - Scientific Coord.)