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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. [email protected]

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CONTRIBUTIONS OF ART TECHNOLOGY IN NEW THERAPEUTIC APPROACHES

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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

[email protected]


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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.


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Therapeutic medicine

S.I.S.ME.R.

VISION 2000

- Gamete and tissue cryo-banking

- PGD – HLA matching

- Stem cells

ART


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Therapeutic medicine

S.I.S.ME.R.

VISION 2000

- Gamete and tissue cryo-banking

ART


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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)


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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


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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


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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


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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


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OOCYTE CRYO-BANKING

OVARIAN TISSUE FREEZING

S.I.S.ME.R.

VISION 2000


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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).


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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.


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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


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OOCYTE CRYO-BANKING

OOCYTE FREEZING – SISMER EXPERIENCE

S.I.S.ME.R.

VISION 2000


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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


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Therapeutic medicine

S.I.S.ME.R.

VISION 2000

- Gamete and tissue cryo-banking

- PGD – HLA matching

ART


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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


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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.


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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)


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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.


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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


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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.


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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.


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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.


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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.


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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)


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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


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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)


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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


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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


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Reproductive Genetics Institute [email protected]

Overall Results and Outcome of Preimplantation Diagnosis for Single Gene Disorders & Preimplantation HLA testing

RGI Experience

04/14/2008


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Therapeutic medicine

S.I.S.ME.R.

VISION 2000

- Gamete and tissue cryo-banking

- PGD – HLA matching

- Stem cells

ART


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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


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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


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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


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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


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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


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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


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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


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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


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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


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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


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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)


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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


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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).


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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


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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)


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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


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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.)


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S.I.S.ME.R.

VISION 2000


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RGI’s Repository of Human Embryonic Stem Cells

S.I.S.ME.R.

VISION 2000

Reproductive Genetics Institute

As of 10/2008


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S.I.S.ME.R.

VISION 2000

CONCLUSIONS:

Therapeutic medicine

- Gamete and tissue cryo-banking

- PGD – HLA matching

- Stem cells

ART


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S.I.S.ME.R.

VISION 2000

CB Stem Cells

CB Stem Cells

Low quantity

High quantity

Direct treatment

In vitro Expansion

Complete engraftment

Locatelli, unpubblished


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