1

Large Scale culture

2

APPLICATIONSAPPLICATIONS

Basic BiologyBasic BiologyCancer BiologyCancer BiologyGenomicsGenomicsDrug DiscoveryDrug DiscoveryCell TherapyCell Therapy

CHARACTERIZATIONCHARACTERIZATION

CD Surface AntigensCD Surface AntigensChemokine ReceptorsChemokine ReceptorsCytokinesCytokinesMitogensMitogensImmunological FeaturesImmunological Features

SOURCESOURCE

Bone MarrowBone MarrowAdipose TissueAdipose TissueUmbilical CordUmbilical CordAmniotic FluidAmniotic FluidSkeletal MuscleSkeletal Muscle& Others& Others

ESC/iPSCESC/iPSC

Multi-lineage Adult Stem Multi-lineage Adult Stem Cells Cells (i.e. (i.e. MAPC, MPLC, MIAMI, etc.)MAPC, MPLC, MIAMI, etc.)

MESODERM MESODERM DIFFERENTIATIONDIFFERENTIATION

EXPANSIONEXPANSIONISOLATIONISOLATION

ChondrocytChondrocyte e

(cartilage)(cartilage)

OsteocytOsteocyte (bone)e (bone)

Adipocyte Adipocyte (fat)(fat)

Myoblasts Myoblasts (muscle)(muscle)

TRANSDIFFERENTIATIONTRANSDIFFERENTIATION

NeuralNeuralHepaticHepaticEndothelialEndothelial

TenocytTenocyte e

(tendon(tendon))

Mesenchymal Stem Cells

3

Human MSC Therapeutic ApplicationsHuman MSCs have become of interest for clinical application due to:

• Capacity for homing and engraftment

• Wide-range differentiation potential

• Immunosuppressive attributes

Potential MSC Therapies:

• Graft versus Host Disease

• Crohn’s Disease

• Bone Defects/ Genetic Disease

• HSC Transplantation

• Cardiac repair

• NIH Clinical Trials search for mesenchymal stem (stromal) cells = >90 studies (www.clinicaltrials.gov)

4

Overview of MultiStem® Production Process

Lot Release & Product Characterization Testing

SterilityPotency

Identity and ViabilityStable Cytogenetics

Absence of tumorigenic potential in vivo

5

MultiStem – Adherent Adult Stem Cell Platform-Athersys

IND Protocol Title Key elements

Phase I, Multicenter, Dose-Escalation Trial Evaluating Maximum-Tolerated Dose of Single and Repeated Administration of Allogeneic MultiStem® in Patients with Acute Leukemia, Chronic Myeloid Leukemia, or Myelodysplasia

IV-delivered productPhase I: open label – SAD, MADAdjunctive to BM/HSC transplant

Phase I, Multicenter, Dose-Escalation Trial Evaluating the Safety of Allogeneic AMI MultiStem® in Patients with Acute Myocardial Infarction

Catheter-delivered productPhase I: open label (w/ registry)

Phase I, Multicenter, Dose-Escalation Trial Evaluating the Safety of Allogeneic MultiStem® in Patients with Ischemic Stroke

IV-delivered productPhase I: blinded, placebo controlled

6

The processing of human MSCs for therapeutic application should be a standardized GMP process, including defined:

(a) Starting material: donor, tissue origin, harvesting, enrichment method, etc.

(b) Tissue culture processing methods: culture medium, culture conditions, etc.

(c) Devices for cell culture: closed (or nearly closed) cell culture system

(d) Quality control:

> input material (donor) analysis

> active components or impurities

> expanded cell karyotype

Human MSC Therapeutic Applications

Sensebé and Bourin Transplantation. 2009 May 15;87(9 Suppl):S49-53. Review.

7

Chondrogenesis

0

1

2

3

4

5

Act

inC

ytos

kele

ton

Sig

nalin

gC

ell C

ycle

:G

1/S

Che

ckpo

int

Eph

rinR

ecep

tor

Sig

nalin

g

ER

K/M

AP

KS

igna

ling

GM

-CS

FS

igna

ling

Inte

grin

Sig

nalin

g

JAK

/Sta

tS

igna

ling

p38

MA

PK

Sig

nalin

g

PD

GF

Sig

nalin

g

PP

AR

Sig

nalin

g

PT

EN

Sig

nalin

g

TG

F-β

Sig

nalin

g

Wnt

/β-

cate

nin

Sig

nalin

g

-lo

g(p

-val

ue) C07

C14

Osteogenesis

0

0.5

1

1.5

2

2.5

3

3.5

Dea

thR

ecep

tor

Sig

nalin

gA

xona

lG

uida

nce

Sig

nalin

g

ER

K/M

AP

KS

igna

ling

Leuk

ocyt

eE

xtra

vasa

tion

Sig

nalin

g

Inte

rfer

onS

igna

ling

Com

plem

ent

and

Coa

gula

tion

p38

MA

PK

Sig

nalin

g

-lo

g(p

-val

ue)

O07

O14

Adipogenesis

0123456789

10

Act

inC

ytos

kele

ton

Sig

nalin

gA

xona

lG

uida

nce

Sig

nalin

gC

ell C

ycle

:G

1/S

Che

ckpo

int

EG

F S

igna

ling

Eph

rinR

ecep

tor

Sig

nalin

g

ER

K/M

AP

KS

igna

ling

Hun

tingt

on's

Dis

ease

Sig

nalin

g

IGF

-1S

igna

ling

Insu

linR

ecep

tor

Sig

nalin

g

Inte

grin

Sig

nalin

g

JAK

/Sta

tS

igna

ling

Leuk

ocyt

eE

xtra

vasa

tion

Sig

nalin

g

Neu

regu

linS

igna

ling

PD

GF

Sig

nalin

g

Wnt

/β-c

aten

inS

igna

ling

-lo

g(p

-val

ue)

A07

A14

* Analysis of active pathways in hMSCs suggests that PDGF, TGFβ and FGF are important signaling pathways for hMSC proliferation and differentiation

7 pathways 13 pathways

15 pathways

Day 14Day 7

3.31

2.82

8.28

2.29

3.46

3.12

0.71

5.38

8.03

9.76

3.4620

4.0910

6.924Tyrphostin (IC50 20 M)

10.772SU 5402 (IC50 10-20 M)

2.8820

6.3510

7.212SB431542 hydrate (IC50 10 M)

1.0140

5.0520

10.38-No inhibitor

Fold increase in viable cell numberConc (M)Inhibitor

Day 14Day 7

3.31

2.82

8.28

2.29

3.46

3.12

0.71

5.38

8.03

9.76

3.4620

4.0910

6.924Tyrphostin (IC50 20 M)

10.772SU 5402 (IC50 10-20 M)

2.8820

6.3510

7.212SB431542 hydrate (IC50 10 M)

1.0140

5.0520

10.38-No inhibitor

Fold increase in viable cell numberConc (M)Inhibitor

Ng et al. Blood. 2008 Jul 15;112(2):295-307 2008

Active Signaling Pathways in Human MSCs

(PDGF inhibitor)

(TGFβR inhibitor)

(FGFR inhibitor)

8

StemPro® MSC SFM Kit (Cat# A10332-01)

Consists of the following:

1. StemPro® MSC SFM Basal Medium (1X)(500ml)

2. StemPro® MSC SFM Supplement (6.67X)(75ml)

Note: Additional components required:

• CELLstart (XenoFree Substrate) (Cat# A1014201)

• GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)

• TrypLE Express (Cat# 12604013) (Animal Origin Free)

StemPro MSC SFM

9

Total Cell Expansion: STEMPRO MSC SFM

0.0E+00

1.0E+06

2.0E+06

3.0E+06

4.0E+06

5.0E+06

6.0E+06

7.0E+06

8.0E+06

9.0E+06

0 1 3 5 7 10

Passage

Net

Tot

al C

ell N

umbe

r P

er F

lask

DMEM + 10% MSC-Qualified FBS

STEMPRO MSC SFM

Adipocyte (Oil Red O)

Chondrocyte

(Alcian Blue)

Osteoblast (Alkaline Phosphase)

0

200

400

600

800

1000

1200

1400

1600

1800

SCM D+PBT D D+P D+B D+T D+PB D+PT D+BT

Growth Factor Supplementation

Rel

ativ

e F

lou

resc

ence

Un

its

(RF

U)

StemPro MSC SFMDMEM + 10% FBS

P = PDGF-BB

B = bFGF

T = TGFβ1

Exp

ansi

on

Dif

fere

nti

atio

n

input cells = passage 5 human Bone Marrow MSCs (4-donor pool)

StemPro MSC SFM

Beadchip Gene Array Analysis

Chondrocyte (Toluidine Blue)

Chase et al. 2009 Submitted.

10

Ste

mP

ro M

SC

SF

MM

EM

alp

ha

+ 1

0%

FB

S

H&E staining; Data published in Agata et al. Biochem Biophys Res Commun 2009;382(2):353-8

Beta Test Data – Dr. Hideaki Kagami – The University of Tokyo

in vivo ectopic bone assay

Experimental Observations:

1) Enhanced primary culture efficacy (more cells faster)

2) Lower alkaline phosphatase activity in undifferentiated cells

3) Greater responsiveness to osteogenic induction

4) Confirmed in vivo ectopic bone formation

Primary Culture

StemPro MSC SFM

11

• A cGMP serum-free medium specially formulated for the growth and expansion of human mesenchymal stem cells (MSCs).

• Contains xenogenic components not amenable to most clinical applications

• Need for a second generation product: StemPro MSC SFM XenoFree

PERFORMANCE - BETTER CELLS - CONVENIENCEPERFORMANCE - BETTER CELLS - CONVENIENCE

StemPro MSC SFM

12

StemPro MSC SFM XenoFree Kit (Cat# A11409SA)(Custom cGMP)

Consists of the following:

1. StemPro® MSC SFM Basal Medium (Cat# A10334-01) (500ml)(1X)

2. StemPro® MSC SFM Supplement (Cat# 09-0012SA) (5ml)(100X)

Note: Additional components required:

• CELLstart (XenoFree Substrate) (Cat# A1014201)

• GlutaMAX (Cat# 35050) or L-Glutamine (Cat# 25030)

• TrypLE Express (Cat# 12604013) or TrypLE Select (Cat# 12563011) (Animal-Origin Free)

Complete cGMP XenoFree workflow: (1) Medium; (2) Substrate; (3) Harvest enzyme

For order inquiries please contact: Sandy Kuligowski (sandra.kuligowski@lifetech.com) or Lucas Chase (lucas.chase@lifetech.com)

StemPro MSC SFM XenoFree

13

StemPro MSC SFM XenoFree

Pas

sa

ge

1

DMEM + 10% MSC-Qualified FBS

Pas

sa

ge

9

Input cells = passage 5 human Bone Marrow MSCs (4-donor pool)

Adipocyte (Oil Red O)

Chondrocyte (Alcian Blue)

Osteoblast (Alkaline Phosphase)

Passage 5 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents

Exp

ansi

on

an

d D

iffe

ren

tiat

ion

StemPro MSC SFM XenoFree: BM-MSC Expansion

StemPro

MSC SFM XenoFree: Doubling Time(Human BM-MSC)

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9

Passage

Do

ub

ling

Tim

e (H

ou

rs)

DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree

StemPro

MSC SFM XenoFree: Net Population Doublings(Human BM-MSC)

0

2

4

6

8

10

12

14

16

18

20

Set-up 1 2 3 4 5 6 7 8 9

Passage

Net

Po

pu

lati

on

Do

ub

ling

s

DMEM + 10% MSC-Qualified FBS StemPro MSC SFM XenoFree

14

MarkerPassage 5

(% Positive)Passage 9

(% Positive)CD73+/NEG- 99.3 99.9CD90+/NEG- 96.4 100.0CD105+/NEG- 96.5 99.9CD34+ 0.1 0.6

MarkerPassage 5

(% Positive)Passage 9

(% Positive)CD73+/NEG- 99.7 100.0CD90+/NEG- 97.9 100.0CD105+/NEG- 98.1 100.0CD34+ 0.2 2.3

StemPro MSC SFM XenoFree

DMEM + 10% MSC-Qualified FBS

NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.

Negative markers-rPE

CD

105-

Ale

xa F

luo

r® 7

00

Negative markers-rPE

CD

73-P

erC

P

CD90-FITC

Ne

ga

tive

mar

kers

-rP

E

CD90-FITC

CD

34-Q

do

t ®

800

Passage 5Passage 5 Passage 9Passage 9

Cells analyzed at Passage 5 and 9 = 46, XY= NORMAL KARYOTYPECells analyzed at Passage 5 and 9 = 46, XY= NORMAL KARYOTYPE

Mu

ltip

lex

Flo

w C

yto

me

try

Jolene Bradford – Life Technolgies Jolene Bradford – Life Technolgies

Kar

yoty

pe

An

alys

is

Minimal Defining Criteria*:(1)Adherence to plastic under standard culture conditions (10% FBS-containing medium)

(2) Characteristic Surface marker expression

Positive (≥95%) Negative (≤2%)

CD73 CD11b or CD14

CD90 CD34

CD105 CD45

CD79a or CD19

HLA-DR

(3) In vitro tri-lineage mesodermal differentiation (osteoblasts, chondrocytes, adipocytes)

*Dominici et al. 2006

StemPro MSC SFM XenoFree: BM-MSC Characterization

15

Multi-Parameter Human MSC Characterization

FxCycle™ Violet stainDNA content

Click-iT® EdU Alexa Fluor® 647 azideCell proliferation

Qdot® 800CD34

HLA-DR

CD45

CD19R-PE

CD14

Alexa Fluor® 700CD105

FITCCD90

PerCPCD73

FluorophoreMarker

CD90-FITCCD

10

5-A

lexa

Flu

or®

70

0

99.6%

0.4%

CD90-FITC

Neg

ativ

e m

ark

ers

-rP

E

100%

CD90-FITCC

D3

4-Q

do

t® 8

00

100%

CD90-FITC

CD

73

-Pe

rCP 99.5%

0.5%

Flow cytometry combiningClick-iT® EdU proliferation analysisWITH

Antibody-based immunophenotypingSimultaneous testing for percentage proliferation with phenotype characterization

Click-iT EdU®

Bright fieldHoechstClick-iT® EdU

HumanMSCs

Clic

k-iT

® E

dU

A

lexa

Flu

or®

64

7 a

zid

e

FxCycle™ Violet DNA

21.5%

FxCycle™ Violet DNA content

Proliferating Human MSCs detected with Click-iT® EdU reagents

16

StemPro MSC SFM XenoFree: BM-MSC Characterization

R2 = 0.910

DMEM + 10% FBS - P3

SP

MS

C S

FM

XF

- P

3

R2 = 0.957

DMEM + 10% FBS – P3

DM

EM

+ 1

0% F

BS

– P

9

R2 = 0.949

SP MSC SFM XF - P3S

P M

SC

SF

M X

F –

P9

NOTES:

• Input cells = P5 MSC (4-donor pool)

• P3 = 13 days in culture

• P9 = 42 days in culture

• Beadchip = HumanWG-6 v3.0

17

StemPro MSC SFM XenoFree OSTEOBLAST

Oil Red O (Day 14)

Alcian Blue (Day 14)

Alkaline Phosphase (Day 14)

Alizarin Red S (Day 21)

OSTEOBLASTCHONDROCYTEADIPOCYTE

Exp

ansi

on

(P

3)

Dif

fere

nti

atio

n (

P3)

StemPro MSC SFM XenoFree: BM-MSC Primary Isolation

MarkerPassage 3

(% Positive)

CD73+/NEG- 99.8CD90+/NEG- 99.9CD105+/NEG- 99.7CD34+ 0.4

StemPro MSC SFM XenoFree - Primary Isolation

NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.

StemPro MSC SFM XenoFree: Primary Expansion(Human BM-MSC)

0.0E+00

2.0E+06

4.0E+06

6.0E+06

8.0E+06

1.0E+07

1.2E+07

0 1 2 3Passage

Ne

t C

ell

Nu

mb

er

Pe

r F

las

k

StemPro MSC SFM XenoFree

Human Serum Human Serum RemovalRemoval

Passage 3 Multi-lineage Mesoderm Differentiation - StemPro Differentiation Regents

18

Human ADSC – SP MSC SFM XF

Shayne Boucher – Life Technologies

StemPro MSC SFM XenoFree: ADSC Expansion

NOTES:

• Input cells = passage 4 StemPro Human ADSCs

• Differentitaion = Passage 3, Day 14 – StemPro Differentiation Reagents

Adipocyte (Oil Red O) Chondrocyte (Alcian Blue)Osteoblast (Alk Phos)

StemPro

MSC SFM XenoFree: Doubling Time(Human ADSC)

0

20

40

60

80

100

120

140

1 2 3 4 5

Passage

Do

ub

ling

Tim

e (

Ho

urs

)

Control Medium StemPro MSC SFM XenoFree

StemPro

MSC SFM XenoFree: Net Population Doublings (Human ADSC)

0

1

2

3

4

5

6

7

8

9

10

Set-up 1 2 3 4 5

Passage

Ne

t P

op

ula

tio

n D

ou

blin

gs

Control Medium StemPro MSC SFM XenoFree

19

StemPro MSC SFM XenoFree: Comparisons

NOTE: input cells = passage 5 Human BM-MSCs (4-donor pool)

Exp

an

sio

n

(Pas

sa

ge

3)

Net Population Doubling: Competitor Audit

0

2

4

6

8

10

12

14

16

18

Set-up 1 2 3 4 5 6 7

Passage

Net

Po

pu

lati

on

Do

ub

ling

s

10% MSC-Qualified FBS StemPro MSC SFM XenoFree Competitor X

P3 Competitor X

CD90-FITC

CD

105-

Ale

xaF

luo

r® 7

00

MarkerPassage 3

(% Positive)CD73+/NEG- 98.8CD90+/NEG- 100.0CD105+/NEG- 100.0CD34+ 0.3

MarkerPassage 3

(% Positive)CD73+/NEG- 98.4CD90+/NEG- 77.3CD105+/NEG- 100.0CD34+ 0.4

StemPro MSC SFM XenoFree

Competitor X

NOTE: NEG = multiplex analysis of CD14, CD19, CD45 and HLA-DR.

StemPro MSC SFM XenoFree Competitor X

Ch

on

dro

cy

te

(Alc

ian

Blu

e)

(Pas

sa

ge

3)

P3 StemPro MSC SFM XenoFree

CD90-FITC

CD

105-

Ale

xaF

luo

r® 7

00

Flow cytometry data generated by Jolene Bradford – Life Technologies

20

Expand Qualify StoreIsolate

Growth factors & substrates

ELISA

Cell selectionbeads

Cell imaging

Gene expression

Media, supplements& single use

processing systems

Cryopreservationmedia

Flow cytometry

Monitor

Dissociation enzymes

Processingdevices

Cell therapy work flow

HLA typing

Immuneresponse

monitoring

21

Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität BochumData generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum

The detailed MSC isolation and expansion workflow was employed to provide an autologous MSC product from human bone marrow aspirate under completely closed conditions using xeno-free cell culture reagents.

ClosedClosed System Xeno-Free MSC Isolation and Expansion

1. 2. 3. 4. 5.

MSC expansion using the

CLINIcell cassetteand StemPro

MSC SFM XenoFree Medium

BM aspirate

Mononuclear cell isolation using

the Sepax system MSC harvest and wash

using TrypLE Select and theCytoMate Cell

Washer

AutologousMSC

product

22Can achieve manufacturing runs equivalent to >200,000L of liquid media

23

Closed system considerations

Media bag ONLY, not a bioreactor!

Pre-filled, stocked and ready when you need them (reduced lead time)

Closed system, ideal for aseptic processes/applications

Small Volume ”Pillow”-Universal Bags” [5L, 10L & 20L]

Large Volume Bags” [100L ,200L, 500L & 1000L]

Easily customized to integrate with

automated systems and process applications (connection methods, in-line filtration, flow rates, tubing lengths, re-circulation loops, etc.)

Conveniently designed for bench top to larger-scale applications

Ideal for ‘daisy-chaining’ set-up

New ‘9101’ PE Film

Old PE Film

(Stedim & Crestbury)

New ‘9101’ PE Film

EVA Film

24

25

Bioreactor – Single use bioreactors

Prefilled Wave O-Series Cellbag

• Ready to ship - media formulations include Sf-900II and Freestyle 293• Convenient - Cellbag chambers are filled and ready to use• Customizable - you choose the GIBCO medium formulation and volume

Size Working Volume

Cellbag10L/O 0.5L – 5LCellbag20L/O 1L – 10L

Connection Methods

• Quick Connect (MPC)• SCD Tubing• Threaded Luers• UNIVERSAL BAGS!!!!!

26

Processing using Dynal® ClinExVivo™ MPC

Primary magnet

Cell depletion bag

Secondary magnet

Sample collection

bag

Sample bag

27

DYNAL® Magnetic Beads

Combining magnetic particles and single use cell expansion technologies

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.

Hollyman D. et al. J Immunother. 2009 Feb-Mar;32(2):169-80.

Phase I Clinical Trial underway at Memorial Sloan Kettering

28

• Undifferentiated hESCs are efficiently depleted from the sampleUndifferentiated hESCs are efficiently depleted from the sample• Higher purity of differentiated cells Higher purity of differentiated cells

• Differentiated cells remain unaffected after purificationDifferentiated cells remain unaffected after purification

Dynabeads® SSEA-4

Dynabeads® SSEA-4 are designed to remove undifferentiated human embryonic stem cells from a culture, providing you with highly pure and differentiated stem cells for your translational applications

Differentiated Neural Stem Cells before and after depletion of SSEA-4+ cells

SS

EA

-4 A

PC

0.2 %14 %

SS

EA

-4 A

PC

29

Von Kossa (Calcium Deposit)

Alkaline Phosphatase

Alcian Blue (Proteoglycan)

Oil Red O (Lipid Vessible)

Osteogenesis

Osteogenesis

Chondrogenesis

Adipogenesis

Day 12 Expanded MSCs

IgG

1

BM

MN

C

IgG1 CD14 CD14/45

IgG

1

CD

90C

D90

CD

105

CD

105

Da

y 12

MS

C

Day 12 expanded mononuclear cells reveals robust selection of CD90 and CD105-positive cells in the absence of hematopoeitic antigen expression (CD14 and CD45), indicative of human MSC expansion. Expanded cells reveal retained multipotency as shown by positive staining results for osteogenesis, chondrogenesis and adipogenesis.

Total Cell Expansion: 3x105 BM MNCs 2x107 MSCs

ClosedClosed System Xeno-Free MSC Isolation and Expansion

Data generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität BochumData generated by Christian Prante, Wolfgang Prohaska and Knut Kleesiek - Ruhr-Universität Bochum

30

Human MSC Culture Systems- Testing

Lot release- pathogen, sterility, identityfunctional quality, viability, consistency

Efficacy tests- Karyotype, epigenome, contaminating cells, HLA type, etc

31

SummarySummary• Therapeutic application of human MSCs will require standardized

GMP-compliant procedures and reagents

• We have developed two generations of cGMP-manufactured serum-free MSC expansion media, including StemPro MSC SFM XenoFree

• Expansion of cells in StemPro MSC SFM XenoFree provides retained MSC phenotype and multipotency with a stable karyotype

• XenoFree medium applied to a closed isolation/expansion protocol reveals a promising clinical culture system

• Continual collaboration will be critical to explore both in vitro and in vivo applications

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

AcknowledgementsRuhr-Universität Bochum (Germany)Ruhr-Universität Bochum (Germany) Christian PranteWolfgang ProhaskaKnut Kleesiek

A*STAR (Singapore)Vivek TanavdeFelicia NgSusie KohKonduru S. R. Sastry

The University of TokyoHideaki KagamiHideki Agata

Case Western Reserve UniversityLuis Solchaga

Life Technologies:Uma LakshmipathyShayne BoucherMohan VemuriSandy KuligowskiDoug DannerSaki PhommachanhJean DonovanMaureen CookBob KendersonAlaine MaxwellKate WagnerMary Lynn TilkinsAndy CampbellJolene BradfordScott Clarke

65