Fibroblast Reprogramming using

Synthetic mRNA
Indra Kusuma
Laboratorium Kultur Sel Univ. YARSI
 Contents

• Induced Pluripotent Stem Cells (iPSCs)

• mRNA Reprogramming
• iPSCs Culture and Characterization
• Embryoid Body Differentiation
• Somatic Cell for reprogramming
 iPSCs
– Induced Pluripotents Stem Cells
• Unlimited Proliferative potential
• Mantain the Capacity to differentiate to any cell type
• Derived from adult patient cells/patient-specific

– Valuable tools for:

• In vitro modelling of human diseases
• Drug discovery
• Unlimited cell source for regenerative medicine
Bernal, 2013
 Cells Reprogramming
– Reprogramming
• Conversion of a somatic/adult cell to the ES-like pluripotent
state
• Morphology, proliferation, gene expression and epigenetics
– Reprogramming strategies
• Somatic Cell Nuclear Transfer (SCNT)
• Cell Fusion
• Direct Reprogramming
– Ectopic expression of transcription factors
– Yamanaka Factors: Oct4, Sox2, Klf4, and c-Myc
– Thomson Factors: Oct4, Sox2, Nanog, Lin28
Hu, 2014
Hu, 2014
 mRNA Reprogramming
– Synthetic/modified mRNA transfected
• Microinjection
• Electroporation
• Liposomal delivery
– Translated mRNA  ectopic expression of
Reprogramming factor (Yamanaka/Thomson)
 Comparison of Method
Delivery Method Pros Cons Efficiency

Retrovirus Lasting Expression Risk of Insetional 0,2%

Mutagenesis
Multiple cell
Low Frequency of division to
Episomal 0,1%
Integration eliminate episomal
DNA
Strong Innate
mRNA Non-Integrating Immunity response 4%
Affected by quality
Absolute non-
rProtein genome integration of recombinant 0,34%
protein
 Comparison of Methode
Delivery Method Pros Cons Efficiency

Retrovirus Lasting Expression Risk of Insetional 0,2%

Mutagenesis
Multiple cell
Low Frequency of division to
Episomal 0,1%
Integration eliminate episomal
DNA
Strong Innate
mRNA Non-Integrating Immunity response 4%
Affected by quality
Absolute non-
rProtein genome integration of recombinant 0,34%
protein
 Comparison of Methode
Delivery Method Pros Cons Efficiency

Retrovirus Lasting Expression Risk of Insetional 0,2%

Mutagenesis
Multiple cell
Low Frequency of division to
Episomal 0,1%
Integration eliminate episomal
DNA
Strong Innate
mRNA Non-Integrating Immunity response 4,4%
Affected by quality
Absolute non-
rProtein genome integration of recombinant 0,34%
protein

Note: Reprogramming using primary

fibroblast results : episomal method 4x Goh et al 2013 & Bernal 2013
retroviral, 50x mRNA
 MACS Academy, 2014

Durruthy et al, 2014

 iPSCs Colonies

MACS Academy, 2014

Isolation of iPS Clones:

• Manual Picking  Cut and Paste Methode Clump passaging

• Enzymatic (TrypLE/Accutase/dispase/collagenase IV)  TRA-1-60 Magnetic

Isolation  Single Cell Passaging (requires ROCK inhibitor)
Cut-Paste Method

MACS Academy, 2014

 iPSCs Characterization
– Morphology of human pluripotent stem cells (hPSCs)
– hPSCs Growth & culture systems
– Spontaneous differentiation
– Alkaline Phospatase staining
– Marker expression – Flow Cyt & ICC
– Karyotyping
– Gene Expression Analysis
– Assesment of Developmental potential:
• In vitro differentiation potential- Embyoid Bodies
• In vivo differentiation potential – teratoma assay
 Human PSCs

Morphology :
• Monolayer culture
• Tightly packed cells
Lab Kultur YARSI, 2014
• Defined colony borders
• High nuclear cytoplasmic ratio
(N/C ratio)

MACS Academy, 2014

 hPSCs Culture systems
– Co-culture systems
• Mouse embryonic fibroblasts (mEF)
• Human foreskin fibroblast (hFF)
– Feeder-free culture
• Matrigel (not Xeno-free)
• Vitronectin
• Laminin
– Serum-free, Xeno-free, Feeder-free culture system
 Spontaneous Differentiation

MACS Academy, 2014

Flat out growing,
No defined colony border
 Alkaline Phospatase staining

MACS Academy, 2014

– Cell membrane enzyme related to pluripotency
– AP= hydrolase enzyme for dephophorylation
– The enzyme hydrolases a substrate which reacts with a colorimetric reagent
and form precipitate
– Red color for undifferentiated AP expressing colonies
 Markers
– Flowcytometry Analysis & Imunocytochemistry
– Reprogramming involves sequential activation of pluripotency
markers
• Alkaline phosphatase
• SSEA-4
• Oct-4 Intracelullar markers
• Nanog
• TRA-1-60 surface markers
– Inconspicuous morphology: TRA-1-60 started downregulating
– Overgrown colonies, flat-out growing: TRA-1-60
downregulated, SSEA-4 started downregulating
 Karyotype of PSCs
– Normal: 46 Chromosomes (44 autosome & 2 sex
Chromosomes); 46,XY
– Abnormal karyotype: 47 chromosomes, trisomy in
chromosome 12; 47,XY+12

– Feeder co-culture is robust and stable: gold

standard
• Gamma/X irradiation : 3000 rad (30 Gy)
• Mytomicin-C: 10ug/ml incubate 2-3 hours, 37oC,5% CO2
 Gene Expression analyis

– Microarray analysis
• DNA-Microarray
• Protein-Array

– Real Time PCR

 Pluripotency Assesment
– In vitro assesment: Embryoid body Formations
– EBs formation (spontaneous differentiation)
• Asses markers form endordermal, mesodermal and
ectodermal lineage
• Imunocytochemistry, Flowcytometry

– In Vivo assesment: Teratoma Assay

• Requires imunodefficient strain animals
• SCID mice or athymic rats
 Embryoid Bodies
– formation of cell aggregates in non-adherent spheroids
– Strategies to induce EB-formation
• Basic methods:
– Hanging drops
– Static suspension
• Alternative methods
– Round bottom
– V bottom
• Bioreactor methods
– Spinner flasks
– Orbital shaker
 3D Differentiation via EBs

Similar to gastrulation of an epiblast-

stage embryo in vivo

Recapiltulates molecular and celullar

morphogenic and inductive signals of
embryo development

Wang et al, 2010

 EBs Dissociation
– EB-derived cells:
• Dissociate Ebs to single cells suspensions at various
stage of differentiation
• Cultivation of EB-derived cells
• Phenotyping or enumeration bt flow cytometry
• Separation of distict cell types using MACS or FACS
– Dissociation
• Efficent dissociation between 4-20 day of EB diff.
• Simple, Trypsinization and manual pippeting (up-down
pippeting/trituration)
 Somatic Cells for Reprogramming
• Patient-spesific cells
– Human Dermal Fibroblasts(HDFs)
– Human Epidermal Keratinocytes (HEKs)
– Peripheral Blood Mononuclear Cells (PBMCs)

Lab Kultur YARSI, 2014

 Fibroblast- YARSI
– Skin eksplant method
– Standard DMEM-10% FBS culture
– Collagen 1 and 3 secretion
– OCT4 mRNA expression
– Cryopreservation ( >80% recovery)
– Doubling Time: 30-40 hours
– Split ratio: 1:10
Keratinocytes Transdifferentiation

Bickenbach, 2013
Chemical Reprogramming

Bickenbach, 2013
 Keratinocytes-YARSI
– Enzymatic Isolation Method
– Serum-free culture system
– Doubling Time: 40-44 hours
– Split Ratio: 1:2

– Currentlly assesed:
• Serum-free cryopreservation
• AQP3 (Aquaporin) mRNA Expression
 PBMCs Reprogramming
– Lentiviral vector & Episomal vector
– CD34+ mononuclear cells (0,01-0,1% in PB)
– CD 33+/CD3-/19- Myeloid /Non Lymphoid
– Magnetic enrichment and serum-free culture
w/EPO

Jun Su et al, 2013

 Conclussions

– Dermal Fibroblast to reprogram

– iPSCs generation using mRNA reprogramming
– Feeder Co-Culture system
– Enzymatic method for single cell passaging
– Embryoid bodies differentiation for in vitro
pluripotency assesment
– Hanging-drop method for EBs formation
Thank You