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

53
© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 Regenerative Medizin: Therapie mit humanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität / Salzburger Landeskliniken Universitätsklinik für Blutgruppenserologie und Transfusionsmedizin S pinal C ord I njury - T issue Re generation C enter S alzburg, SCI-TReCS

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

Page 1: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Regenerative Medizin:Therapie mit humanen

Zellen/Faktoren(Human Cell-Based Medicinal Products)

Eva Rohde, Paracelsus Medizinische Privatuniversität / Salzburger Landeskliniken

Universitätsklinik für Blutgruppenserologie und Transfusionsmedizin

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

Page 2: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

SCI-TReCS Start

Page 3: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

SCI-TReCS

Spinal Cord Injury - Tissue Regeneration Center Salzburg

Page 4: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 5: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 © Paracelsus Medizinische

Privatuniversität | Lehrveranstaltung

PMU / SCI-TReCSBasic Science & Development

Under Constructions (IX/2013)

Page 6: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

•Internationally Recognized Scientific LocationWithin 5 - 10 Years Attracting Further Specialists

•Focus on SCI & Regenerative Medicine•Persisting 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

Page 7: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

SCI-TReCS Mission

Practising Multi-Disciplinarity

Page 8: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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,...

Page 9: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 10: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 11: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

1 x 106 = mouse setting, 10cm2

MesenchymaleStammzellen

MSC110cm

1 x 109 = human setting2.8m2

Page 12: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | 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

Page 13: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

GMP-Requirements (cell products)

Page 14: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Cell Product Characterization

Page 15: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Cell Product Characterization(Authorities)

Page 16: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Process Optimization

Page 17: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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.

Page 18: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 19: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 20: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 Salzburger Landeskliniken

Morphology & Proliferation

Page 21: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 Salzburger Landeskliniken

PHA Response Modulation

Page 22: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 Salzburger Landeskliniken

Serum Supplementation versus Serum-Free Culture

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

Page 23: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Outdated versus Fresh HPL

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

Page 24: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

MSC DetachmentEfficiency & Viability

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

Page 25: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 25

MSC

ECFC

0 min. 24 hrs.

Proliferation & Migration

M. Gimona, Transfusion Medicine Salzburg

Page 26: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 26

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”

Page 27: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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- βVEGF-A, -CPDGFFactor VMultimerinFactor VIIIMMPsα2-Macroglobulin

PlasminogenPAI-1CXCL-5MIP-1αRANTESCCL17IL-8P-SelectinVon Willebrand FactorThrombospondinFibrinogenIntegrin αIIbβ3Integrin αvβ3Fibronectin...............

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 Frühwirth Margret Frühwirth Claudia UrlClaudia UrlRokhsareh RohbanRokhsareh Rohban Birgit FeilhauerBirgit Feilhauer

Dirk StrunkDirk Strunk

Page 28: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Implantation of Cell/Matrix composites subcutaneously in NSG mice

NSG

MSPC/ECFC (20:80)

in Extracellular Matrix

MSPC pure in

Extracellular Matrix

Extracellular Matrix

only (CTRL)

ECFC pure in

Extracellular Matrix

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

Page 29: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 29

Studying The Impact of Platelets on Regeneration

IgGbFGFEGFHGFCTGFIGF-1TGF- βVEGF-A, -CPDGFFactor VMultimerinFactor VIIIMMPsα2-Macroglobulin

PlasminogenPAI-1CXCL-5MIP-1αRANTESCCL17IL-8P-SelectinVon Willebrand FactorThrombospondinFibrinogenIntegrin αIIbβ3Integrin αvβ3Fibronectin...............

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

Page 30: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

- 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?

Page 31: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 31

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

Page 32: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 32

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

Page 33: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 33

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. Öller, K. Schallmoser E. Rohde, Salzburg

Page 34: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 35: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 36: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 36

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

Page 37: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Induced

Induced

+ BM-MSPC Exosomes

Induced

+ UC-ECFC Exosomes

Alzarin Red S staining at day 14 post osteogenic induction

However, exosomes modulate the speed and magnitude of

differentiation

Exosomes & Osteogenic Lineage Induction

Page 38: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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

Page 39: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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 Power

To Cover the Complex Requirements of:

• Quality and Manufacturing Aspects

• Non-Clinical Development

• Clinical Development

Precondition for Clinical Studies to Evaluate Cell-basedTherapies – GMP for RegMed Needs Much Ressources

Transfusion Medicine Provides the Perfect Link!

Page 40: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Transfusion Medicine: M. Gimona M. Öller

K. Schallmoser

D. Streif

T. Lener

S. Laner-Plamberger

D. Peckl-Schmid

Page 41: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Page 42: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Thank You For Your Attention!

We Are Also Grateful to:

Paracelsus Medical University Salzburg

Salzburger Landeskliniken

„Wings For Life“ for Catalyzing the SCI-TReCS Project

And: The SCI-TReCS Sponsor!

Page 43: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | SISCIC Consortium

Page 44: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | SISCIC Consortium 44

Calponin & Actin at 20x magnification

Calponin & Actin at 100x magnification

Contractile vSMC / pericytemarkers in MSCs

M. Gimona, Transfusion Medicine Salzburg

Page 45: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | SISCIC Consortium

DANKE FÜR IHRE AUFMERKSAMKEIT!

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

Universitätsklinik für Blutgruppenserologie und Transfusionsmedizin der PMU Salzburger Landeskliniken Lindhofstraße 20-22 A - 5020 Salzburg

STEM CELL RESEARCH UNIT GRAZ

Page 46: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | 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

Alzarin Red S staining at day 14 post osteogenic induction

Page 47: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013 47

Paxillin (top), pSrcY416 (bottom)& Actin

Mechanosensory molecules and mechanoresponse in UC-MSCs

Cortactin (top), ILK (bottom) & Actin

untreated 500 nM PDBu 500nM Y-27632

Salzburger Landeskliniken

Mechanosensation & - Response

M. Gimona

Page 48: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

DDR2 in BM-MSCs at P1

Phalloidin DDR2

Rb anti DDR2 extracellular discoidin domain

Mo anti DDR2 intracellulardomain

Phalloidin DDR2

Salzburger Landeskliniken

M. Gimona

Page 49: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

GMP-requirements (facility)

!!!GMP-Berater!!!

Page 50: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | SISCIC Consortium

Page 51: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Präklinische

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

Salzburger Landeskliniken

Page 52: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

Potential GMP-Labor

GMP-Labor

SISCIC

PMU

Ermöglicht Translationale Forschung:Klinische Anwendung Zelltherapie Industrie-Kooperationen

Zelluläre ToxizitätstestsPharmakologische Entwicklung

Kräftiger Impuls für R & D „Klinische Zelltherapie“

in Salzburg

Herstellung innovativer Therapien

Intellectual PropertyVerwertung

Effizienz der Verwendung und Focus? Salzburger Landeskliniken

Page 53: Regenerative Medizin: Therapiemithumanen … Medizin: Therapiemithumanen Zellen/Faktoren (Human Cell-Based Medicinal Products) Eva Rohde, Paracelsus Medizinische Privatuniversität

© Paracelsus Medizinische Privatuniversität | Salzburg | SCI-TReCS | Rohde | ÖGBT Wien | 7.6.2013

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%

Salzburger Landeskliniken