Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human...

Paracelsus Medizinische Privatuniversitt | Salzburg | SCI-TReCS | Rohde | GBT Wien | 7.6.2013

Regenerative Medizin:Therapie mit humanen

Zellen/Faktoren(Human Cell-Based Medicinal Products)

Eva Rohde, Paracelsus Medizinische Privatuniversitt / Salzburger Landeskliniken

Universittsklinik fr Blutgruppenserologie und Transfusionsmedizin

Spinal Cord Injury - Tissue Regeneration Center Salzburg, SCI-TReCS


SCI-TReCS Start


SCI-TReCS

Spinal Cord Injury - Tissue Regeneration Center Salzburg


SCI-TReCS Mandate

SCI-related clinical research projects

Providing best state of the art treatment and care of patients with spinal cord injury (SCI)

Developing novel integrative therapeutic strategies for tissue regeneration and functional repair after organ injury


Internationally Recognized Scientific LocationWithin 5 - 10 Years Attracting Further Specialists

Focus on SCI & Regenerative MedicinePersisting Multidisciplinary Efforts Should Result in SuccesfulBasic Science Output & Long-term Clinical Projects

Years 1 3Establishing SCI-TReCSDesign of 1stClinical Trials Years 3 5

ConsolidationStarting withClinical Trials? Beyond Year 5

Ongoing & Emerging Clinical TrialsIPR Royalties, 3rd Party-Financing New Partners & Sponsors

SCI-TReCS Road Map


SCI-TReCS Mission

Practising Multi-Disciplinarity


SCI-TReCS Approach

Practising Multi-Disciplinarity Basic Research

(Discovery) Translation Application Patient Care

GMP-Facility / Transfusion Medicine

Experimental & Clinical Cell Therapy

Experimental Neuro-Regeneration

Bone & Tendon Regeneration

Molecular Regenerative Medicine

Clinical Wards: Traumatology, Neurology, Urology,...

Neuro-Surgery, Rehabilitation Centers, Nursing Sciences,...


SCI-TReCS ConstructSpinal Cord Injury and Tissue Regeneration Center

Salzburg (SCI-TReCS)Organigram Draft Version 1.0

International Scientific Advisory Board

Local Scientific Steering Committee

SCI-TReCS Operating Director(TBA) Administartive & Coordinating Support

for Scientific Units and Core Facilities

GMP-

Facil ity

Small

Animal

Imaging

Animal

Surgery

Micro-

scopy

Flow

cytometry

Core Facilities:

Preclinical Scientific UnitsStrubergasse

Exp. SCI &

Preclinical

Neuro-Rehab..

Clinical &

Experimental

Cell Therapy

Molecular

Regenerative

Medicine

Bone &

Tendon

Regeneration

Neuro-

ImagingNeuro-

Rehabilitation

Pelvic Floor

Neurostimul-

ation

Clinical Scientific UnitsHospital wards

Acute

Surgery

Neurophysi-

ology and

Paraplegiology

HistologyMolecular

Biology

Bio-

Informatics

-Statistics

Bio-

Hazard


Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS)

Organigram Draft Version 5.0

Local Scientific Steering Committee(6 members: H. Resch, M. Nake, L. Aigner, G. Janetschek, E. Trinka, E. Rohde)

GMP-

Facil ity

Core Facilities:

Preclinical Scientific Units (Location: Research Center Strubergasse)

Neuro-

Surgery

Anaesth-

esiology

Ophthalm-

ology

Clinical Scientific Units (Location: Clinical

Deptartments)

Dermat-

ology

Sponsor-Committee(2-3 members: Financial Sponsor, PMU)

2x /Year SCI-TReCS Progress Report

Local User Board

Existing

AdministrativePMU Infrastructure

furthercl inical

cooperat.

Depts.

further

cl inicalcooperat.

Depts.

2x / Year Update/Progress Report

* red:

future units/structures

to be established

Neur-

ology

Urology &Pelvic FloorNeuromod.

Acute &

Trauma

Surgery

Trans-

fusion

Medicine

Radiology

International Scientific Advisory Board(6 - 7 members: TBA)

SCI-TReCS Managing

CoordinatorAdministrative Support

for Scientific Units and Core Facilities

Small

Animal

Imaging

Animal

SurgeryMicro-

scopy

Flow

cytometry

Exp. SCI &

Neuro-

Regeneration

Clinical &

Experimental

Cell Therapy

Molecular

Regenerative

Medicine

Bone &

Tendon

Regeneration

HistologyMolecular

Biology

Bio-

Hazard

Bio-

Informatics

-Statistics

Annual

SCI-TReCS Day

ASCIS

CTC


1 x 106 = mouse setting, 10cm2

MesenchymaleStammzellen

MSC110cm

1 x 109 = human setting2.8m2

Paracelsus Medizinische Privatuniversitt | SISCIC Consortium

Defined GMP-Requirements

Personell Responsibilities

Quality Training / Awareness

Continous Improvement

Defined Lab Conditions

Defined Climate Condition

Process validation

Product licence (IMPD)

CT, Ethical Issues

Documentation / 30y traceability

Facilities

Human Cell-basedMedicinal Products (=pharmaceuticaldefinition)

Salzburger Landeskliniken


GMP-Requirements (cell products)


Cell Product Characterization


Cell Product Characterization(Authorities)


Process Optimization


Optimization II

1000/cm2

day 1

day 3

day 5

day 10

100/cm2 10/cm2 1/cm2

P+1 P+1

Figure 12.3

Inverse Correlation of Seeding Density to MSC Proliferation.


Choice of SupplementsFigure 12.4

MSC Proliferation Capacity Depends on Seeding Density in Xenogeneic FBS and Human

Platelet Lysate-supplemented Cultures.

0

2

4

6

8

10

1/cm2 10/cm2 100/cm2

FBS HPL

FBSFBS HPLHPL

1/cm1/cm22 1/cm1/cm22

10/cm10/cm22 10/cm10/cm22

100/cm100/cm22 100/cm100/cm22

0

200

400

600

800

1/cm2 10/cm2 100/cm2

FBS HPL

PD

(d 1

3)P

D (d

13)

Fo

ld in

crea

seF

old

incr

ease

MSC cultured for 13 days MSC cultured for 13 days


Identity/PurityPhenotype Characterization

HLA-AB CD 13 CD 29 CD 73 CD 90 CD105 CD 146

CD 45 CD 3HLA-DR CD 31CD 14 CD 31CD 34

FBS

FBS

HPL

HPL

GD-2

Figure 12. 5

Immune Phenotype of Human MSC.

CD 19

100 101 102 103 104CD133-APC

100 101 102 103 104CD133-APC

CD 133


PHA Response Modulation


Serum Supplementation versus Serum-Free Culture

Animal Protein-Free Expansion of human MSPCs (In: Regenerative Therapy using Blood-Derived Stem Cells, Springer 2012


Outdated versus Fresh HPL

Salzburger LandesklinikenAnimal Protein-Free Expansion of human MSPCs (In: Regenerative Therapy using Blood-Derived Stem Cells, Springer 2012


MSC DetachmentEfficiency & Viability

Salzburger LandesklinikenAnimal Protein-Free Expansion of human MSPCs (In: Regenerative Therapy using Blood-Derived Stem Cells, Springer 2012


00,5

11,5

22,5

33,5

44,5

1

Ce

ll In

de

x

without collagen with exosomes

without collagen without exosomes

0

0,51

1,5

2

2,53

3,5

4

1

Ce

ll I

nd

ex

with collagen with exosomes

with collagen without exosomes

Proliferative and Migratory Capacity

M. Gimona & T. Lener, Transfusion Medicine Salzburg

xCELLigence System, Impedance Measurement Real-time Cell-based Assays


UC-MSPCs in pHPL

MSPCs ECFCs HPL ExosomesMesenchymal Endotheliale Human mRNA/miRNA/ProteinStem- and Progenitor Colony Forming Platelet Lysate Containing MicrovesiclesCells Cells

100% 80%20%

IgGbFGFEGFHGFCTGFIGF-1TGF- VEGF-A, -CPDGFFactor VMultimerinFactor VIIIMMPs2-Macroglobulin

PlasminogenPAI-1CXCL-5MIP-1RANTESCCL17IL-8P-SelectinVon Willebrand FactorThrombospondinFibrinogenIntegrin IIb3Integrin v3Fibronectin...............

Osteogenesis / Regeneration

Neovasculogenesis

Basic Research

STEM CELL STEM CELL RESEARCH UNITRESEARCH UNITMedical University of Graz, AustriaMedical University of Graz, Austria

Andreas Reinisch Andreas Reinisch Christina BartmannChristina BartmannNicole Hofmann Nicole Hofmann Katharina Schallmoser Katharina Schallmoser Nathalie Nathalie EtchartEtchart Anna OrtnerAnna OrtnerDaniela Thaler Daniela Thaler Monica FarrellMonica FarrellMargret Frhwirth Margret Frhwirth Claudia UrlClaudia UrlRokhsareh RohbanRokhsareh Rohban Birgit FeilhauerBirgit Feilhauer

Dirk StrunkDirk Strunk


Implantation of Cell/Matrix composites subcutaneously in NSG mice

NSG

MSPC/ECFC (20:80)

in Extracellular Matrix

MSPC pure in

Extracellular Matrix


only (CTRL)

ECFC pure in


Autologous MSPC / ECFC pairs Inject in to NSG mouse

6.4 million:25.6 million (20:80)

MSC-ECFC signaling in vivo:Modulating Neovasculogenesis?

18-24 h

2 week 8 weeks

plaque explantation

early signalling eventschecked with antibody microarray

accepted in: PLOSONE 2013R. Rohban, E. Rohde, D. Strunk, Graz-Salzburg


Studying The Impact of Platelets on Regeneration

IgGbFGFEGFHGFCTGFIGF-1TGF- VEGF-A, -CPDGFFactor VMultimerinFactor VIIIMMPs2-Macroglobulin

PlasminogenPAI-1CXCL-5MIP-1RANTESCCL17IL-8P-SelectinVon Willebrand FactorThrombospondinFibrinogenIntegrin IIb3Integrin v3Fibronectin...............

Schallmoser K, Strunk D Basic Cell Culture Protocols, Humana Press, 2013

Revealing the Active Substances of Pooled Human Platelet Lysate, pHPL

Cell Culture / Differentiation / Regeneration


- the microenvironment decisively modulates cell fate

- cells contribute to their own microenvironment (ECM, soluble factors, microvesicles)

Thus, the differentiation of stem and progenitor cells can be seen as a consequence of at least three regulatory processes:

1) Mechanotransduction (response to ECM, stiffness etc.)

2) Growth factor receptors (response to the receptor mediated induction of signal transduction cascades)

3) Exosomes (response to intracellularly delivered RNA species and proteins)

30

How Do (Endothelial and Mesenchymal) Cells Interact?


Co

nce

ntr

atio

n

Nanoparticle Tracking Analysis (NTA)

CD9

23kDa

17kDa

CD63

50kDa

33kDaParticle size ~ 30-100nm

Mean: 130nm +/- 18nm

Characterization of Stem Cell Exosomes

ExosomeWestern Blots

Exosomes are mRNA/miRNA/Protein containing small vesicles

delivering Molecular Cargo between cells


Purification of stem cell exosomes from conditioned media

Conditioned Culture Medium

Supernatant #1

Supernatant #2

Pellet 1(discarded)

10 min @ 2.000 x g

Pellet 2(discarded)

20 min @ 20.000 x g

90 min @ 120.000 x g

Pellet 32 x wash and centrifugation @ 120.000 x g

Supernatant #3 (discarded)

Exosomes used for experiments


Osteo-Induction (+HPL) + BM-MSC exosomes + ECFC exosomes

Adipo-Induction + BM-MSC exosomes + ECFC exosomes

Studying Osteogenesis- / Adipogenesis-Modulation with Exosomes

Manuscript in preparationM. Gimona, T. Lener & D. Peckl-Schmid, D. Streif, M. ller, K. Schallmoser E. Rohde, Salzburg


Uptake of heterotypic stem cell exosomes

BM-MSPCs UC-ECFCs

34

UC-ECFC ExoBM-MSPC Exo

Immunofluorescence microscopy images

red: Alexa 568 phalloidingreen: PKH67

BM-MSPC with UC-ECFC exosomes UC-ECFC with BM-MSPS exosomes


Heterotypic Exosomes Increase Proliferation (xCELLigence Analyses)

0

0,2

0,4

0,6

0,8

Ce

ll In

de

x

0,0

0,5

1,0

0

1

2

3

4

5

Ce

ll In

de

x

0,0

0,1

0,2

0,3

BM-MSPC - +Exosomes

BM-MSPC - +Exosomes

UC-ECFC - +Exosomes

UC-ECFC - +Exosomes

Ce

llco

un

t[m

io]

BM-MSPC UC-ECFC

Ce

ll c

ou

nt

[mio

]

0 0

Cell culture conditions: alpha MEM + 10% human platelet lysate (HPL)Cell index values determined by impedance measurements using the xCELLigence System from ACEA after 3 days in cultureCell counting performed after 14 days in culture

B C D


Exosomes & Osteogenic Lineage Induction

Alzarin Red S staining at day 14 post osteogenic induction

Uninduced

Uninduced + BM-MSPC (homotypic) Exosomes

Uniduced + UC-ECFC (heterotypic) Exosomes

Exosomes do NOT induce differentiation


Induced

Induced

+ BM-MSPC Exosomes

Induced

+ UC-ECFC Exosomes


However, exosomes modulate the speed and magnitude of

differentiation

Exosomes & Osteogenic Lineage Induction


Uninduced Induced Adipogenic induction Adipogenic induction+ BM-MSPC Exosomes + UC-ECFC Exosomes

(homotypic) (heterotypic)

0

20

40

60

80

100

120

140

1

uninduced

induced

induced + bmmsc exos DA001

induced+ecfc exos 0036

noninduced

induced

induced + BM-MSPC exosomes

induced + UC-ECFC exosomes

%

Exosomes & Adipogenic Lineage Induction

Sudan 3 staining at day 14 post osteogenic induction


RegMed Conclusion

Development of Human Cell-based Medicinal Products for Regenerative Therapies Needs:

Personell Practising Pharmazeutikal QM Production Facilities / Clinical Environment Basic Science Facilities / Brain PowerTo Cover the Complex Requirements of:

Quality and Manufacturing Aspects Non-Clinical Development Clinical DevelopmentPrecondition for Clinical Studies to Evaluate Cell-basedTherapies GMP for RegMed Needs Much Ressources

Transfusion Medicine Provides the Perfect Link!


Transfusion Medicine: M. Gimona M. ller

K. Schallmoser

D. Streif

T. Lener

S. Laner-Plamberger

D. Peckl-Schmid


Thank You For Your Attention!

We Are Also Grateful to:

Paracelsus Medical University Salzburg


Wings For Life for Catalyzing the SCI-TReCS Project

And: The SCI-TReCS Sponsor!

Paracelsus Medizinische Privatuniversitt | SISCIC Consortium 44

Calponin & Actin at 20x magnification

Calponin & Actin at 100x magnification

Contractile vSMC / pericytemarkers in MSCs

M. Gimona, Transfusion Medicine Salzburg


DANKE FR IHRE AUFMERKSAMKEIT!

Salzburger Landeskliniken Eva Rohde, Univ.-Klinik fr Blutgruppenserologie und Transfusionsmedizin

Universittsklinik fr Blutgruppenserologie und Transfusionsmedizin der PMU Salzburger Landeskliniken Lindhofstrae 20-22 A - 5020 Salzburg

STEM CELL RESEARCH UNIT GRAZ

Paracelsus Medizinische Privatuniversitt | SISCIC Consortium 46

Induced

Induced+ BM-MSPC Exosomes

Induced+ UC-ECFC Exosomes

Sustained matrix deposition of induced & replated BM-MSPCs

BM-MSPC Exosomes

without Exosomes

Osteogenic lineage induction of BM-MSPC



Paxillin (top), pSrcY416 (bottom)& Actin

Mechanosensory molecules and mechanoresponse in UC-MSCs

Cortactin (top), ILK (bottom) & Actin

untreated 500 nM PDBu 500nM Y-27632


Mechanosensation & - Response

M. Gimona


DDR2 in BM-MSCs at P1

Phalloidin DDR2

Rb anti DDR2 extracellular discoidin domain

Mo anti DDR2 intracellulardomain

Phalloidin DDR2


M. Gimona


GMP-requirements (facility)

!!!GMP-Berater!!!


Prklinische

Entwicklung

PMU

Forschung

GMP-Facility

PMU et al

Pharma-

Entwicklung

Pharma

LKH & CDK

Versorgung

Rehabilitat.

Partner/

Experten

Forschung

national

Forschung

international

Patient

Salzburg Interdisciplinary Spinal Cord Injury Centre - SISCIC



Potential GMP-Labor

GMP-Labor

SISCIC

PMU

Ermglicht Translationale Forschung:Klinische Anwendung Zelltherapie Industrie-Kooperationen

Zellulre ToxizittstestsPharmakologische Entwicklung

Krftiger Impuls fr R & D Klinische Zelltherapie

in Salzburg

Herstellung innovativer Therapien

Intellectual PropertyVerwertung

Effizienz der Verwendung und Focus? Salzburger Landeskliniken


Potential use for a GMP-Lab?

GMP-Labor

SISCIC

PMU

SCI Knochen/Sehnen

MuskelNeuro

Augen/Netzhaut

Haut

Effizienz der Verwendung und Focus?

SCI Neuro Muskel Augen Haut Knochen/Sehnen

~ 70%


Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human...

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