EX VIVO GENERATION AND EXPANSION OF MESENCHYMAL STEM CELLS FOR CLINICAL APPLICATION Pediatric Haemat

1
EX VIVO GENERATION AND EXPANSION OF MESENCHYMAL
STEM CELLS FOR CLINICAL APPLICATION Pediatric
Haematology/OncologyIRCCS Policlinico San
Matteo, Pavia, Italy
2
PROPERTIES of MESENCHYMAL STEM CELLS (MSCs)

• Multipotent cells
• capable of differentiation into several
  mesenchymal lineages
• Enhancement of hematopoietic stem cell
  engraftment
• Immunosuppressive properties
• Tissue reconstitution

3
MULTILINEAGE POTENTIAL OF MSCs (1)

• Potential to differentiate into lineages of
  mesenchymal
• tissues
• bone - fat- cartilage - tendon - muscle

Pittinger MF et al. Science 284 143-147,
1999
4
MULTILINEAGE POTENTIAL OF MSCs (2)

• Isolation of multipotent mesenchymal stem cells
  from umbilical cord blood.
• O.K. Lee et al. Blood 2004 1031669-1675.
• Ectodermal and endodermal differentiation MSCs
  are also able to differentiate into neuroglial-
  and hepatocyte-like cells

5
PROPERTIES of MESENCHYMAL STEM CELLS (MSCs)

• Multipotent cells
• capable of differentiation into several
  mesenchymal lineages
• Enhancement of hematopoietic stem cell
  engraftment
• Immunosuppressive properties
• Tissue reconstitution

6
ENHANCEMENT OF HSC ENGRAFTMENT

• It has been shown in varoius animal models that
• co-tranplantation of MSCs enhances engraftment
• of hematopoietic stem cells
• Noort W. et al. Exp. Hematology (2002)
  mice
• MSCs promote engraftment of human UCB-derived
  CD34 cells
• in NOD/SCID mice
• El Badri NS et al. Exp. Hematology (1998)
  mice
• Almeida-Porada G. et al. Blood (2000)
  sheep

7
Co-Tx of fetal lung-derived MSCs enhances
engraftment of UCB CD34 cells in NOD/SCID mice

Noort W. et al. Exp. Hematology 2002 30 870-878
8
PROPERTIES of MESENCHYMAL STEM CELLS (MSCs)

• Multipotent cells
• capable of differentiation into several
  mesenchymal lineages
• Enhancement of hematopoietic stem cell
  engraftment
• Immunosuppressive properties
• Tissue reconstitution

9
IMMUNOREGULATORY PROPERTIES (1)

• LOW IMMUNOGENICITY of MSCs
• low expression of costimulatory molecules
• no stimulation of lymphocyte proliferation in
  mixed lymphocyte cultures (MLC)
• escape of lysis by cytotoxic lymphocytes and NK
  cells
• suppression of T cell responses
• Interaction of human mesenchymal stem cells
  with cells involved inalloantigen-specific
  immune response favors the differentiation of
  CD4 T-cell subsets expressing a
  regulatory/suppressive phenotype
• Maccario R et al. Haematologica
  200590516-525

10
IMMUNOREGULATORY PROPERTIES (2)

• Expanded MSCs co-infused with HLA-identical
  hemopoietic stem cell transplants reduce acute
  and chronic GVHD a matched pair analysis.
• Frassoni F et al. EBMT 2002
• MSC Controls
• N. of pts 31 31
• BM/PBSC 17/14 17/14
• MSCs 1-2 x 106/Kg 0
• Engraftment 31 31
• Acute GVHD I 9 13
  p0,002
• II 5 10
• III-IV 2 5
• Chronic GCHD 32 11 67 10
  p0,02
• Survival at 6 months 96 4 68 8
  p0,02

11
IMMUNOREGULATORY PROPERTIES (3)

• Treatment of severe acute graft-versus-host
  disease with third party haploidentical
  mesenchymal stem cells
• Le Blanc K et al. The Lancet 2004 363 1439-1441

12
PROPERTIES of MESENCHYMAL STEM CELLS (MSCs)

• Multipotent cells
• capable of differentiation into several
  mesenchymal lineages
• Enhancement of hematopoietic stem cell
  engraftment
• Immunosuppressive properties
• Tissue reconstitution

13
TISSUE RECONSTITUTION

• Potential clinical use of MSCs in regenerative
  medicine and tissue engineering
• repair of skeletal tissues
• myocardial repair
• CNS or spinal injury
• MSCs are an attractive source for such a purpose
  because of their pluripotency and availability

14
SOURCES OF MSCs

• Adult bone marrow
• Periosteum
• Trabecular bone
• Muscle connective tissue
• Fetal tissues (bone marrow, lung, liver, spleen)
• Amniotic fluid and placenta
• Umbilical cord blood

15
Rationale for using MSCs in HSCT recipients

• To reduce the risk of GVHD in HLA-identical
  sibling HSCT
• To reduce the risk of GVHD in UD HSCT
• To improve hematopoietic engraftment in CBT
  recipients
• To improve immune reconstitution in CBT
  recipients
• To improve immune reconstitution in T-cell
  depleted HLA-partially matched HSCT
• To treat steroid-refractory GVHD.

16
CLINICAL APPLICATIONS OF MSCs IN HSC
TRANSPLANTATION

• HLA-identical Tx
• Unrelated Tx
• UCB Tx
• Haplo-identical Tx
• GVHD treatment

17
MSC EXPANSION PROTOCOL

• The protocol has been developed in
• LEIDEN UNIVERSITY MEDICAL CENTER
• by Dr. H. Roelofs and Prof. W. Fibbe and
• KAROLINSKA INSITUTE, Stockolm
• by Dr. K. Le Blanc
• MSCs are isolated and ex vivo expanded under GMP
  conditions to numbers required for clinical
  application
• (1-2 x 106 cells/Kg recipient weight)
• and injected I.V. at 4 hours before the infusion
  of HSCs

18
MSC SOURCE for CLINICAL APPLICATION

• BONE MARROW
• (50 ml)
• harvested 6 weeks before Tx
• in local anesthesia under sterile conditions
• and collected in sterile heparin-containing tubes

19
MATERIALS

• REAGENTS
• DMEM-Low Glucose
• Penicillin/Streptomycin (P/S)
• Fetal Calf Serum (FCS) from selected
  batches
• Trypsin/EDTA
• Culture medium DMEM-LG - P/S - 10 FCS
• Washing/Collection medium DMEM-LG - P/S - 2 FCS
• Tissue culture flask T25-T75-T175

20
SOP 1 ISOLATION OF MNCs FROM B.M.

• Dilution and filtration of BM sample
• Density separation (Ficoll 1,077 g/ml) 20
  minutes at 1000 G, RT
• Washes 10 minutes at 350 G, RT
• Cell count and viability in a Bürker chamber

21
SOP 2 INITIATION OF IN VITRO MSC EXPANSION

• Plating of MNCs in culture flasks to initiate
  the in vitro MSC expansion
• MNCs are resuspended in culture medium and
  plated at
• 160.000 cells/cm2
• in tissue culture flasks
• Flasks are placed in the incubator at 37C, 5 CO2

22
SOP 3 CELL CULTURE CHECK AND SAMPLE COLLECTION

• Inspection of the cultures twice a week (every
  3-4 days) for
• - contamination
• - confluency and morphology
• Collection of samples for sterility
  (aerobe/anaerobe) and Mycoplasm
• pool medium from several flasks

23
CONFLUENCY and MORPHOLOGY

• CONFLUENCY
• the cells need to be replated when the flasks
  are approximately 70 confluent
• MORPHOLOGY
• spindle-shaped

24
SOP 4 REFRESH

• Replace culture medium of in vitro expanding MSCs
  with fresh medium
• TWICE a WEEK (every 3-4 days)

25
SOP 5 TRYPSINIZE

• Wash the adherent cells with PBS
• Add Trypsin/EDTA and incubate at 37 C
• for 5 minutes
• Add collection medium to stop the Trypsin
  reaction and bring the cells in suspension
• Centrifuge 10 minutes, 350 G, RT
• Counts cell concentration and viability
• REPLATE (SOP 6)
• Do not handle more than 5 flasks at a time

26
SOP 6 REPLATE

• Reseed the trypsinized MSCs in culture medium for
  further expansion
• MSCs are resuspended in culture medium and
  replated
• (PASSAGED) at
• 4.000 cells/cm2
• in tissue culture flasks
• Place flasks in the incubator at 37C, 5 CO2

27
SOP 7 HARVEST

• Harvest the expanded MSCs for
• administration (SOP 8) or
• cryopreservation (SOP 9)
• Wash the adherent cells with PBS
• Add Trypsin/EDTA and incubate at 37 C for 5
  minutes
• Add harvest medium and bring the cells in
  suspension
• Centrifuge 10 minutes, 350 G, RT
• Resuspend the cells in administration buffer and
  count
• Do not handle more than 5 flasks at a time

28
SOP 8 PREPARATION FOR ADMINISTRATION

• Calculate the cell dose and volume of cell
  suspension to be administered
• Transfer the cell suspension to be administered
  to a transfer bag (CLOSED SYSTEM) that will be
  carried to the unit
• Administration of the cells to the patients
• Take a sample for cryopreservation

29
ADMINISTRATION BUFFER

• The administration buffer contains
• saline solution
• serum proteins
• and NO FCS
• that has been washed out during the harvest
  procedure.
• The cells are resuspended in a final
  concentration of 2 x 106 cells/ml

30
SOP 9 CRYOPRESERVATION

• Cryopreserve cell product for future
  administration
• transfer the cell suspension to a freezing
  container (CLOSED SYSTEM)
• add cryopreservation mix (20 DMSO)
• freezing procedure in a programmable cryovessel
• store cell product in a CLINICAL N2-tank

31
SOP 10 ADMINISTRATION AFTER CRYOPRESERVATION

• Prepare cell product for administration after
  cryopreservation
• calculate the volume of cell product to be thawed
• thaw cell product in a water bath
• add 50 v/v NaCl 4 C and mix gently
• transfer of cell product to the unit and
  administration to the patient

32
SAMPLE COLLECTION

• FACS analysis
• T1 on bone marrow sample pre-Ficoll
• T2 at 11-8 days pre-Tx
• T3 at harvest (day Tx)
• STERILITY (aerobe/anaerobe)
• T1 on supernatant post-Ficoll
• T2 at 11-8 days pre-Tx
• T3 at harvest (day Tx)
• MYCOPLASM
• T1 on supernatant post-Ficoll
• T2 at 11-8 days pre-Tx
• T3 at harvest (day Tx)

33
QUALITY CRITERIA for RELEASE of IN VITRO EXPANDED
MSCs

• FACS analysis
• gt 90 of the cell population positive for CD
  73-90-105
• lt 10 of the cell population positive for
  HLA-DR CD 31- 34- 45- 80
• Sterility (including Mycoplasm)
• Morphology spindle-shape
• Aspect colorless cell suspension devoid of cell
  aggregates

34
PANEL for IMMUNOPHENOTYPE

• Antigen
• HLA-I
• CD 73 (SH3/SH4)
  positive
• CD 90 (Thy-1)
• CD 105 (Endoglobulin)
• CD 34
• CD 45
• HLA-II
  negative
• CD 80
• CD 31 (PECAM)

35
DECISION TREE
Harvest BM
Ficoll separation
Initiation of MSC expansion
Check cultures at 2-4 days/Refresh medium
Check cultures every 3-4 days
gt70 confluency
lt70 confluency
Trypsinize/Replate
Refresh medium
Check confluency every 3-4 days
Trypsinize/Replate
Harvest
Refresh
Cryopreseration
Prepare for administration
RELEASE
Administration after cryo
36
USE of FETAL CALF SERUM

• To date in vitro expansion of MSCs is obtained
  in the presence of
• 10 FETAL CALF SERUM (FCS)
• Corcerns related to the use of FCS
• possible infection transmission (viruses,
  bacteria, prions)
• immune or local inflammatory reactions due to
  contamination with bovine proteins
• Regulatory restrictions exist on the use of FCS
  in several countries

37
SUBSTITUTES FOR FCS

• Efforts are directed towards developing FCS-free
  media
• AUTOLOGOUS SERUM
• Autologous serum for isolation and expansion
  of human mesenchymal stem cells for clinical use.
  Stute N. et al. Exp. Hematology 32 (2004)
• PLATELET-RICH PLASMA (PRP)
• - Platelets are a natural source of growth
  factors
• - PRP is an autologous concentration of human
  platelets in a small volume of plasma

38
Acknowledgements

• DEPARTMENT OF EXPERIMENTAL HEMATOLOGY
• Leiden University Medical Center
• Dr. A. J. Nauta
• Dr. H. Roelofs
• Prof. W. Fibbe