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Aronowitz stem cell 11.25.2010
1. Stem Cell Update 2010
Joel A. Aronowitz, MD
University Stem Cell Center
Cedars Sinai Medical Center
USC, Division of Plastic Surgery
2. Stem Cell Update
• Scientific basis of ADSC use
• Separation methods
• FDA position
• Recap of IFATS Dallas, Oct’10
• Applications in clinical practice
Adipose Derived Stem Cell
Labeled flourescent antibody
for “CD 34+” cell marker
4. liver stem cells
• Heart -Heart - rare! only 1 in 40,000 heart cellsrare! only 1 in 40,000 heart cells
cardiac stem cell
brain stem cell
Stem cells found in virtuallyStem cells found in virtually
all organsall organs
kidney stem cells
• Brain -Brain - rare, found in subventricular zonerare, found in subventricular zone
• Kidney -Kidney - no good markers for stem cellsno good markers for stem cells
• Pancreas -Pancreas - very hard to find (2008)very hard to find (2008)
pancreas stem cell
• Intestines -Intestines - found in base of cryptsfound in base of crypts
intestinal stem cells
• Lung -Lung - may also be the cells that formmay also be the cells that form
lung cancer (2006)lung cancer (2006)
lung stem cells
• Hair -Hair - found in base of hair folliclesfound in base of hair follicles
Pluripotential Cells
5. Stem Cell ComparisonsStem Cell Comparisons
Comparing 6 Stem Cell Types
FeatureFeature Embryonic
harvest embryo Bone m. blood skin lipo no skin bx
method destruction aspiration draw biopsy or BM
potency high mod mod high mod high
can form yes no no yes no no
teratomas??
culture yes yes yes yes no yes
required?
gene no no no yes no no
transfer
required?
Muse Cells
7. Advantages of Adipose Stem Cells
• Adipose tisue is rich in stem cells, >20%
of cells vs 0.5 to 0.1% in bone marrow,
cardiac < 0.001%.
• Separation possible at “Point of Service,”
ie OR. Harvest 50,000 cells from
marrow vs 5-50 million stem cells from
adipose.
• Adipocyte survival, <24 hrs, <30%
ADSC survival >72 hrs, >70%.
• High metabolic rate vs anoxic tolerance
of pluripotential cells, stem cells
stimulated by injury.
8. • CBS 60 minutes: 1st expose’ on stem cell scam - Stowe
Biotherapy
• Offshore SC clinic proliferation - now over 30 clinics
worldwide
• 1st deaths from IV autologous, cultured, adipose derived SC
therapy.
• FDA does not approve 510K application by Cytori for Cellution
machine.
2010 Negative SC News
9. • 1st ESC clinical trial started
• CIRM 1.3 billion allocated to date
• Worldwide clinical trials ~ 2,500 with stem cells being
conducted worldwide
• 800+ cases of ASC-enriched fat grafting done worldwide
• 50 clinical trials with fat derived stem cells being conducted
worldwide, None in US.
• MUSE cells identified in fat.
2010 Positive SC News
10. Muse Stem
Cells
Muse Stem
Cells • Can be cultured in “Can be cultured in “floating culturefloating culture” technique” technique
• Can differentiate into endodermal, ectodermal,Can differentiate into endodermal, ectodermal,
& mesodermal cells in vitro and in vivo& mesodermal cells in vitro and in vivo
• HomingHoming - will home into damaged skin, muscle,- will home into damaged skin, muscle,
or liver w/IV injectionor liver w/IV injection
• Differentiate into appropriate cells when injectedDifferentiate into appropriate cells when injected
into skin, muscle, or liver by local or IV injectioninto skin, muscle, or liver by local or IV injection
• Contain “Contain “SSEA-3+SSEA-3+” protein” protein
• Not as high proliferation activity as ESCsNot as high proliferation activity as ESCs
• Can be cultured in “Can be cultured in “floating culturefloating culture” technique” technique
• Can differentiate into endodermal, ectodermal,Can differentiate into endodermal, ectodermal,
& mesodermal cells in vitro and in vivo& mesodermal cells in vitro and in vivo
• HomingHoming - will home into damaged skin, muscle,- will home into damaged skin, muscle,
or liver w/IV injectionor liver w/IV injection
• Differentiate into appropriate cells when injectedDifferentiate into appropriate cells when injected
into skin, muscle, or liver by local or IV injectioninto skin, muscle, or liver by local or IV injection
• Contain “Contain “SSEA-3+SSEA-3+” protein” protein
• Not as high proliferation activity as ESCsNot as high proliferation activity as ESCs
MMultilinageultilinage SStresstress EEndurancendurance CCellsells
Dezawa, M, et. al, PNAS, April 26, 2010
11. • ADSCs secrete growth factorsADSCs secrete growth factors - FGF, HGF,VEGF, PDGF, PDGF, etc.
• Growth Medium determines ADSC differentiationGrowth Medium determines ADSC differentiation - FBS vs HS- FBS vs HS
• ADSCs can form form fatADSCs can form form fat - when stimulated with neuropeptideY
• ADSCs can form cardiac myocytesADSCs can form cardiac myocytes - when grown in M3534
• ADSCs can form endothelial cellsADSCs can form endothelial cells - when growth medium for
endothelial cell formation is used
• ADSCs secrete growth factorsADSCs secrete growth factors - FGF, HGF,VEGF, PDGF, PDGF, etc.
• Growth Medium determines ADSC differentiationGrowth Medium determines ADSC differentiation - FBS vs HS- FBS vs HS
• ADSCs can form form fatADSCs can form form fat - when stimulated with neuropeptideY
• ADSCs can form cardiac myocytesADSCs can form cardiac myocytes - when grown in M3534
• ADSCs can form endothelial cellsADSCs can form endothelial cells - when growth medium for
endothelial cell formation is used
Meeting Highlights - Basic ScienceBasic Science
8th Annual IFATS Meeting
Dallas, TX 10/22-24, 2010
*IFATS=International Federation of
Adipose Therapeutics and Science
12. • ADSCs can be grown w/no FBSADSCs can be grown w/no FBS - no xenogenic proteins
• ADSCS grow on Hyaluronic acidADSCS grow on Hyaluronic acid - A new scaffold material for tissue
engineering and facial soft tissue augmentation (Restylane)
• Adipose tissue can be decellularized and used as a scaffoldAdipose tissue can be decellularized and used as a scaffold - like
acellular dermal grafts (Alloderm)
• ADSC can grow on HydrogelsADSC can grow on Hydrogels - A new scaffold material - not toxic to
adipose derived stem cells
• ADSCs can be grown w/no FBSADSCs can be grown w/no FBS - no xenogenic proteins
• ADSCS grow on Hyaluronic acidADSCS grow on Hyaluronic acid - A new scaffold material for tissue
engineering and facial soft tissue augmentation (Restylane)
• Adipose tissue can be decellularized and used as a scaffoldAdipose tissue can be decellularized and used as a scaffold - like
acellular dermal grafts (Alloderm)
• ADSC can grow on HydrogelsADSC can grow on Hydrogels - A new scaffold material - not toxic to
adipose derived stem cells
Meeting Highlights- Basic Science
8th Annual IFATS
Conference
13. • ADSCs heal partial thickness burns - Mouse model
• ADSCs heal chronic wounds - Porcine model
• ADSCs improve cartilage graft survival - Rat model
• ADSCs improve cardiac function in MI - Porcine model
• ADSCs endothelialize vascular grafts - mechanism -VEGF
• ADSCs improve pancreatic islet cell survival - Mouse model effect
probably due to cytokine secretion by ADSCs
• ADSCs heal partial thickness burns - Mouse model
• ADSCs heal chronic wounds - Porcine model
• ADSCs improve cartilage graft survival - Rat model
• ADSCs improve cardiac function in MI - Porcine model
• ADSCs endothelialize vascular grafts - mechanism -VEGF
• ADSCs improve pancreatic islet cell survival - Mouse model effect
probably due to cytokine secretion by ADSCs
Meeting Highlights - Animal Studies
8th Annual IFATS
Conference
14. • High density fat better than low density fat - bottom layer of fat
(after centrifugation) has more stem cells
• Low negative pressure syringe aspiration - better than high negative
pressure syringe aspiration of fat survival
• Large volume fat grafting to breast safe - 250cc volume increase in
breast with one fat grafting session using Brava device, pre and post op
(No ADSC enrichment)
• 3D imaging and MRI show 2X increase in breast size - with fat
grafting alone - NO ADSC enrichment
• High density fat better than low density fat - bottom layer of fat
(after centrifugation) has more stem cells
• Low negative pressure syringe aspiration - better than high negative
pressure syringe aspiration of fat survival
• Large volume fat grafting to breast safe - 250cc volume increase in
breast with one fat grafting session using Brava device, pre and post op
(No ADSC enrichment)
• 3D imaging and MRI show 2X increase in breast size - with fat
grafting alone - NO ADSC enrichment
Meeting Highlights - Fat Grafting
8th Annual IFATS Conference
15. • Rubin &Yoshimura -Rubin &Yoshimura - Mammographic post CAL breast augmentationMammographic post CAL breast augmentation
compared to post breast reductioncompared to post breast reduction
• lower rate of mammographic abnormalities post CAL
• Higher rate of mammographic abnormalies after reduction
• Yoshimura –Yoshimura – most CAL proceduresmost CAL procedures - 521 patients treated in Japan
• average volume for breast aug - 100-300cc
• Complications - oil cysts, calcifications, pneumothorax
• Rubin &Yoshimura -Rubin &Yoshimura - Mammographic post CAL breast augmentationMammographic post CAL breast augmentation
compared to post breast reductioncompared to post breast reduction
• lower rate of mammographic abnormalities post CAL
• Higher rate of mammographic abnormalies after reduction
• Yoshimura –Yoshimura – most CAL proceduresmost CAL procedures - 521 patients treated in Japan
• average volume for breast aug - 100-300cc
• Complications - oil cysts, calcifications, pneumothorax
Meeting Highlights - CAL Human Studies
8th Annual IFATS Conference
Thanks to James Watson, MD for IFATS update
16. Old Theory Emerging Theory
“It’s the Cells”
Stem Cells divide
and differentiate
into cells needed for
tissue/organ repair
and regeneration
“It’s the Cytokines”
Trophic factors: over
800 proteins are secreted
ADSC’s. Systemic & local
effects, direct regeneration
of tissue and “crosstalk”
between cells.
New Evidence of Adipose StemNew Evidence of Adipose Stem
Cell Function in LipotransferCell Function in Lipotransfer
18. Manual separation inManual separation in
OROR
Fat cells are
harvested via
liposuction
ADS’s isolated with
Multistation,
60-90min
Fat & ADSC’s
Injected
into the patient
19. 2. After centrifuge
Fat layer
Free oil & Tumescent &
RBC layer
1. 1000G, 3~5 minutes
4. Pure fat without free oil
tumescent fluid and RBC
3. Remove Free oil &
tumescent fluid & RBC
layer (A part)
After removing free
oil(upper part) remove
tumescent and RBC
A
SAL with standarized
protocol
20. 1.Mixing Collagenase
Solution with pure fat
Collagenase
Solution
pure fat
2.After hand shaking,
put it in incubator at
37 , 200 rpm, 30 min.℃
3. Fat after Shaking
incubation
21. Lipoaspirate 500 cc, 400 cc processed
Cell count; 2.05 million cells/ml
Injection; 30 cc fat + 10 million cells, viability 83%
22. Study data sheet
Patient C188176
Patient Data
Age: 75 Weight: 200lbs Height: 6’1
H/O Radiation: No
Meds/Steroids: No
Allergies: Keflex/ PCN
BMI prior to liposcuction: 26.4
Chief complaint: unhealing wound left inner ankle- 1 year
Location: Left inner ankle
Pre-op photo: Yes
Fat Harvest Data
Harvesting area: abdomen
Estimation of fat thickness “pinch test”: 2 cm
Tumescent fluid: Hunstad formula
Volume of tumescent: 300cc
Lipoaspirate volume prior to centrifugation: 60cc
Use Centrifugation in OR: Yes
Lipoaspirate volume after washing, centrifugation: 50cc
Lipoaspirate quality: Yellow
Est. harvesting time-prior to processing: 20 min
23. Study data sheet
Patient C188176
Patient Data
ADSC Isolation Data
Starting volume of fat: 60cc
Centrifugation: 10 minutes/ 800 G
Collagenase incubation: 30 minutes/ 200 shaker RPM
Collagenase neutralization washing #washing cycles: 3
Total time to isolate stem cells: 90 minutes
Final volume of stem cell: 8cc resuspended
Photo of stem cell isolate: Yes
Cell Data
Cell density: 100,000 cells/cc
Total cell count (est. –cc x cell density): 800,000 cells
CD34+ labeling : No
Procedure Data
Area ADSC’s were injected: left ankle
Injection technique: 20g needle + 100cc syringe
Volume injected: 8cc
Overcorrection: No
Infiltration with : .25% Marcaine Plain
Injection w/ PRP(Platelet Rich Plasma): No
Other procedures done concurrently: No
Post Op Patient Data
Post Op complications: None
Wound healing insitu-days: 30 days
Wound healing donor site-days: 14 days
24. ADSC enhanced STSG of
chronic wound
S.G 73 yo woman with left
distal tibial wound, non
healing for over 22 mo., 6
months care in Tower
Wound Care Center.
43. FDAFDA andand Stem CellsStem Cells
(The FDA may view Stem Cells as a New Drug)(The FDA may view Stem Cells as a New Drug)
• 1997 -1997 - FDA announces plansFDA announces plans for regulation of CT & CTBP - 62 FR 9261for regulation of CT & CTBP - 62 FR 9261
• 1998 -1998 - Manufacturers registration -Manufacturers registration - allall CT & CTBPCT & CTBP
manufacturers must register and be listed @ FDA - 63 FR 26744manufacturers must register and be listed @ FDA - 63 FR 26744
• 2001-2001- Product registrationProduct registration - all human- all human CT & CTBPCT & CTBP must bemust be
registered with the FDA - 21 CFR 207, 208, & 1271registered with the FDA - 21 CFR 207, 208, & 1271
• 2004 -2004 - Donor eligibilityDonor eligibility - FDA forms regulation for eligibility of- FDA forms regulation for eligibility of
donors of CT & CTBP - 21 CFR 210, 211, 820donors of CT & CTBP - 21 CFR 210, 211, 820
cGTP final rulescGTP final rules - FDA forms “Current Good Tissue Practic” rules- FDA forms “Current Good Tissue Practic” rules
for CT & CTBPs - 21 CFR 16, 1270, 1271for CT & CTBPs - 21 CFR 16, 1270, 1271
Donor screeningDonor screening - FDA forms regulation for screening, testing,- FDA forms regulation for screening, testing,
and related labeling of all CT & CTBP - 21 CFR 1271and related labeling of all CT & CTBP - 21 CFR 1271
• 2005 -2005 - FDA decides to regulate stem cells as a drugFDA decides to regulate stem cells as a drug
• 2010 -2010 - Legal challengeLegal challenge of FDA’s jurisdiction over stem cell therapy made byof FDA’s jurisdiction over stem cell therapy made by
Regenexx, a Colorado based SC company - now on lawsuit #3Regenexx, a Colorado based SC company - now on lawsuit #3
From Drugs to Cells:From Drugs to Cells: History of “Regulatory Creep” by FDAHistory of “Regulatory Creep” by FDA
44. Stem Cell ResearchStem Cell Research
Barriers to Clinical Use of Stem Cells in the US
• Regulation of SC culturingRegulation of SC culturing
”cGTP” rules
• Regulation of SC bankingRegulation of SC banking
GMP rules
• Regulation of biologicalRegulation of biological
tissue transporttissue transport - FTC- FTC
• FDA wants to regulateFDA wants to regulate
stem cells as astem cells as a drug - ie- ie
Phase I, II, and III trialsPhase I, II, and III trials
• IRBs in US -- very difficult tovery difficult to
get approval w/o FDAget approval w/o FDA
approvalapproval
• Patent problemsproblems
•delays in patent issuancedelays in patent issuance
•lawsuits over patent infringementlawsuits over patent infringement
• High cost of doing researchof doing research
• Political interferenceinterference
• Ethical objectionsobjections
• Public ignorance - US public- US public
mis/uninformedmis/uninformed
Government Barriers Non-Government Factors
You can see freshly isolated cells under miscroscope,
but many other cells are contaminated such as erythrocytes and leukocytes.
ADSCs are fibroblast like cells after attachment but round before attachment
1-10 billion nucleate cells can be obtained from 1,000 ml aspirated fat and at least 10% of the cells are ADSCs
