Stem Cell Update 2010Joel A. Aronowitz, MDUniversity Stem Cell CenterCedars Sinai Medical Center

USC, Division of Plastic Surgery

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

Types/Sources of “Stem Cells”Types/Sources of “Stem Cells”

• Unfertilized egg (parthenogenesis)• Fertilized egg (embryonic)mbryonic)• Fetal Fetal • Blood DerivedBlood Derived

• Adult peripheral bloodAdult peripheral blood• Umbilical cord bloodUmbilical cord blood

• Placental Placental • Synthetic - iPS cellsSynthetic - iPS cells• MUSE cellsMUSE cells• Tumor Stem CellsTumor Stem Cells

• Tissue Specific• bone marrowbone marrow• fat derivedfat derived• cardiac derivedcardiac derived• brain derivedbrain derived• Liver derivedLiver derived• Blood vessel derivedBlood vessel derived• GI derivedGI derived• Lung derivedLung derived• Tooth derived• Hair follicle derived• Endometrial

Adult Stem Cells Embryonic, & Other Sources

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 Stem cells found in virtually all organsvirtually all organs

kidney stem cells

• Brain -Brain - rare, found in subventricular zone rare, 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 form may 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

Stem Cell Stem Cell ComparisonsComparisons

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

ADSC’sADSC’s

SkeletalSkeletalMuscleMuscle

BoneBone CartilageCartilage

FatFat Liver cellsLiver cells Blood vesselsBlood vessels

Nerve tissueNerve tissueCardiacCardiacMuscleMuscle

AdiposeDerived

Stem Cells

(ADSCs)

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.

• 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

• 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

Muse StemCells

Muse StemCells • 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 injected Differentiate 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 injected Differentiate 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 S Stresstress E Endurancendurance C Cellsells

Dezawa, M, et. al, PNAS, April 26, 2010

• ADSCs secrete growth factorsADSCs secrete growth factors - FGF, HGF, VEGF, PDGF, PDGF, etc.

• Growth Medium determines ADSC differentiation Growth Medium determines ADSC differentiation - FBS - FBS vs HSvs HS

• ADSCs can form form fatADSCs can form form fat - when stimulated with neuropeptide Y

• ADSCs can form cardiac myocytes ADSCs can form cardiac myocytes - when grown in M3534

• ADSCs can form endothelial cells ADSCs 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 differentiation Growth Medium determines ADSC differentiation - FBS - FBS vs HSvs HS

• ADSCs can form form fatADSCs can form form fat - when stimulated with neuropeptide Y

• ADSCs can form cardiac myocytes ADSCs can form cardiac myocytes - when grown in M3534

• ADSCs can form endothelial cells ADSCs 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

• ADSCs can be grown w/no FBS ADSCs 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 Adipose tissue can be decellularized and used as a scaffoldscaffold - 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 FBS ADSCs 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 Adipose tissue can be decellularized and used as a scaffoldscaffold - 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

• 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

• 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

• Rubin & Yoshimura - Rubin & Yoshimura - Mammographic post CAL breast Mammographic post CAL breast augmentation compared to post breast reductionaugmentation compared 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 Mammographic post CAL breast augmentation compared to post breast reductionaugmentation compared 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

Old Theory Emerging Theory

“It’s the Cells”Stem Cells divideand differentiateinto cells needed for tissue/organ repair and regeneration

“It’s the Cytokines”Trophic factors: over800 proteins are secreted ADSC’s. Systemic & local effects, direct regeneration of tissue and “crosstalk” between cells.

New Evidence of Adipose New Evidence of Adipose Stem Cell Function in Stem Cell Function in LipotransferLipotransfer

New Evidence of Adipose New Evidence of Adipose Stem Cell Function in Stem Cell Function in LipotransferLipotransfer

ADSC isolation techniques

• Automated separation

•Manual lab separation

•Culture expansion

•Cryo preservation

• Induction agents/scaffolds

•Non homologous use

Manual separation in Manual separation in OROR

Fat cells areharvested via

liposuction

ADS’s isolated withMultistation,

60-90min

Fat & ADSC’sInjected

into the patient

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

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

Lipoaspirate 500 cc, 400 cc processedCell count; 2.05 million cells/ml

Injection; 30 cc fat + 10 million cells, viability 83%

Study data sheet

Patient C188176Patient 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

Study data sheet

Patient C188176Patient Data

ADSC Isolation DataStarting volume of fat: 60ccCentrifugation: 10 minutes/ 800 GCollagenase incubation: 30 minutes/ 200 shaker RPMCollagenase neutralization washing #washing cycles: 3Total time to isolate stem cells: 90 minutesFinal volume of stem cell: 8cc resuspendedPhoto of stem cell isolate: Yes

Cell DataCell density: 100,000 cells/ccTotal cell count (est. –cc x cell density): 800,000 cellsCD34+ labeling : No

Procedure DataArea ADSC’s were injected: left ankleInjection technique: 20g needle + 100cc syringeVolume injected: 8ccOvercorrection: NoInfiltration with : .25% Marcaine PlainInjection w/ PRP(Platelet Rich Plasma): NoOther procedures done concurrently: No

Post Op Patient DataPost Op complications: NoneWound healing insitu-days: 30 daysWound healing donor site-days: 14 days

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.

Preop ankle ulcer

Stem cell fraction isolated

ADSC Isolation Data

•Starting Volume of Fat 140cc

•Centrifugation: 10 minutes at 800G

•Collagenase incubation 30 min; 200 shaker RPM

Collagenase Neutralization

• Washing-# washing cycles: 4 cycles

• Total Time to Isolate Stem Cell: 90 minutes

• Final Volume of Stem Cell: 8cc Resuspended

Culture Results (-)Aerobic C & S

Cell Data

•Cell Density: 2,050,000 cells/cc. 5 cc

Procedure Data

• Area ADSC’s were injected: Left Lower Leg

• Injection technique: gauge needle 10cc syringe

•Volume injected: 8cc

• Inflitration w/lidocaine w/epi? No

• Injection w/ PRP: No

• Other procedures done concurrently: yes-skin graft

STSG and Injection of stem cell/adipose mixture to wound base

and adjacent tissue

3 weeks postop

• 8-10-10

4 weeks postop

• 8-17-10

6 weeks postop

• 8-31-10

Complete healing, 9 weeks postop

• 9-28-10

45 yo with chronic acne scars, loss of volume

Preop

Cell assisted lipotransfer

Lipoaspirate 500 cc, 400 cc processedCell count; 2.05 million cells/ml

Injection; 30 cc fat + 10 million cells

2 weeks postop

Post Op 5 months Cell assisted lipo transfer

Pre Op

Pre Op Post Op, 5 months ADSC treatment

Open facial wound after basal cell skin cancer

surgery

Left cheek scar and contour defect after skin

closure

Cell assisted fat grafting to facial defect

1 week post op

2 weeks post op

6 months Post op

Post op

FDAFDA and and Stem CellsStem Cells (The FDA may view Stem Cells as a New (The FDA may view Stem Cells as a New Drug)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 - all all CT & CTBP CT & 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 be must 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 by of 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

• 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 - all all CT & CTBP CT & 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 be must 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 by of 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

Umbilcal Cord CellsUmbilcal Cord Cells • GeronGeron• BiotransplantBiotransplant

• GeronGeron• BiotransplantBiotransplant

From Drugs to Cells:From Drugs to Cells: History of “Regulatory Creep” History of “Regulatory Creep” by FDAby FDA

Stem Cell Stem Cell ResearchResearch

Barriers to Clinical Use of Stem Cells in the US

• Regulation of SC Regulation of SC culturing culturing ”cGTP” rules

• Regulation of SC Regulation of SC banking banking GMP rules

• Regulation of Regulation of biological tissue biological tissue transporttransport - FTC - FTC

• FDA wants to regulate FDA wants to regulate stem cells as astem cells as a drug - - ie Phase I, II, and III ie Phase I, II, and III trialstrials

• IRBs in US - - very very difficult to get approval difficult to get approval w/o FDA approval w/o FDA approval

• Patent problems problems•delays in patent issuancedelays in patent issuance•lawsuits over patent lawsuits over patent infringementinfringement

• High cost of doing of doing research research

• Political interference interference• Ethical objections objections • Public ignorance - US - US

public mis/uninformedpublic mis/uninformed

Government Barriers

Non-Government Factors

End…