Introduction•Stemcell-basedregenerativemedicinewillrevolutionizecurrentapproachesandprovidetreatmentsforavarietyofcurrentlyincurablediseases[Weismann 2000].

•Anumberofstemcelltherapiesalreadyexisttoday,suchastreatingbonemarrowtransplantation,regen-erationandhealingofboneinspinalfusionsurgery[Orhtofix],preventionofacuteGraftvsHostDisease[LeBlanc 2008]andmoretreatmentsareonthehorizon.

•Mesenchymalstemcells(MSCs)havebeenleadingthewayinstemcelltherapydevelopment.AsofDecem-ber2010,over160clinicaltrialswithMSCshavebeenreportedforsuchdiseasesasmultiplesclerosis,autoim-munediseases,diabetes,cardiovasculardisease,Crohn’s,cirrhosis,andotherdiseases[www.clinicaltrials.gov].

•Amnioticfluidisemergingasoneofthemostat-tractivesourcesofMSCswithbroadutilityfortissuecreationandregeneration[Kaviani 2001; t’Anker 2003],andgrowthoftrachea,diaphragm,boneandheartvalveshasbeendemonstrated[Kunisaki 2006; Fuchs 2004; Steigman 2009; Schmidt 2008.]

•Currently,amnioticfluidMSCsarecollectedprimarilyduring2ndtrimestergeneticamniocentesis,thuslim-itingitsbroadavailability.Inthisstudy,weexploredwhetheramnioticfluidcollectedduring3dtrimesterisaviablesourceofMSCsfortherapeuticpurposes

•Ithasbeenpreviouslydemonstratedthat3dtrimesteramnioticfluidcontainsMSCswithcharacteristicssimilartoMSCsfrom2ndtrimesterfluid[You 2009]butprotocolsforcollectionshavenotbeenimplementedandcharacterizationofcellsaftercryopreservationhasnotbeenperformed,andarethemainfocusofourresearch.

Stem Cells – Sources

“Mesenchymal stem cells are poised to be the next major success in cell therapy that could be used to treat

tens of thousand of patients.”—Dr. Jeffrey Karp, MIT/Brigham & Women’s,

December 18th, 2009•MSCsarebecomingthestemcellsofchoiceformanynewtherapeuticdevelopment.

•MSCsarepluripotentstemcellsthatareabletodifferentiateintomanytypesofcellsincludingskin,muscle,neurons,cardiactissue,kidney,liver,cartilage,bone,etc,andarepotentiallyusefulforabroadrangeoftherapeuticapplications[Kaviani 2001; de Coppi 2007.]

•MSCswereinitiallyidentifiedinadultbonemarrow,butlaterwerefoundinothertissues,includingamnioticfluid.

Worldwide Human Clinical Trials with MSCs

160+humantrialsareon-goingworld-widewith5,000+patientsenrolled[Ankrum 2010; www.

clinicaltrials.gov]

Amniotic Fluid is an Attractive Source of MSCs

•AmnioticfluidishighlyconcentratedsourceofMSCs[Caplan 2009; Steigman 2009]•Concentration of mesenchymal cells decreases dramatically

with age, from 1/100 in amniotic fluid, to 1/10,000 in umbilical cord blood, to 1/250,000 – 1/2,000,000 in adult bone marrow

•AmnioticfluidMCSsaregeneticallystableandnon-oncogenic[Miranda-Sayago 2011]

•AmnioticFluidMCSsposseshighproliferationcapacity,solargequantitiesofcellscanbegrownoutofsmallinitialsample

Amniotic Fluid is a Unique Source of MSCs For Tissue Engineering

•Amnioticfluidcellsarehighlyamendabletotissueengineering[Fuchs 2004; Kunisaki 2006; Kunisaki 2007]:•Express high levels of elastin•Resistant to hypoxic (low oxygen) conditions•Posses immunomodulation properties

•2-5ccof2ndtrimesteramnioticfluidissufficienttogenerate100+millionofcellsinseveralweeks–sufficienttosupportsurgicaltissuereplacement/graft[t’Anker 2003, Kunisaki 2006.]

•ProtocolstocollectandcryopreservethesecellshavebeendevelopedfortheapprovalbyFoodandDrugAdministration[Kunisaki 2007; Steigman 2008.]

Ethical Consideration•Thereisnocontroversyorethicaldilemmainamnioticfluidstemcellscollectionandusebecausetheircollectiondoesnotharmthefetus.

“...[amniotic fluid] stem cell source is considered morally licit as it does not require the destruction of

human embryos….and is called by many “the future of medicine.

—L’Osservatore Romano, February 2010

“Amniotic-fluid stem cells underscore the advances in so-called “ethical” stem cells, which hold the potential

to revolutionize medical treatment without being contentious.”

—The Washington Times, December 2009

Figure 1. Tissue Grafts Have Been Grown from Amniotic Fluid MSCs.

WithRecentadvancementsintissueengineering,tis-suesandorganpartscanbecreatedinjustfewweeksandutilizedtotreatconditionssuchasdiaphragmatichernia,trachealatrisia,andasternaldevelopment.

Third Trimester Amniotic Fluid as Source of Mesenchymal Stem Cells for Regenerative ApplicationsLucy Bayer-Zwirello1, Dario Fauza2, Myriam Armant3, Renee Procopio4, Emanuela Roselli5, Gaia Zanna1, Massimiliano Manganini4, Kate Torchilin4, Federico Maggi5, Francesca Grati5, Giuseppe Simoni4,5

1St. Elizabeth Medical Center, Boston, MA, 2Children’s Hospital, Surgery, Boston, MA, 3Immune Disease Institute, Center for Human Cell Therapy, Boston, MA, 4Biocell Center, Medford, MA and Varese, Italy, 5TOMA Advanced Biomedical Assays S.p.A, Varese, Italy

Neuron  

Stem  Cells  

Hematopoie.c  stroma  

Tenocyte  

Osteocyte  

Chondrocytes  

Muscle  Pathway  

Neural  Pathway  

Adpocyte  

Skeletal  muscle  

Smooth  muscle  

Cardiac  muscle  

Astrocyte  

Oligodendrocyte  

Mature  Cells  

Mesenchymal    Stem  Cells  

Sources: In’tAnker 2003; Modifiedfrom http://hemo-genix.com/the_mesenchymal_stem_cell_system/

Mesenchymal Stem Cells are Pluripotent

MSC

s pe

r Mar

row

Cel

ls

1

2’000’000

1

400’000

1

250’000

1

100’000

1

10’000

1

100 Human MSCs Decline With Age

7  

Amnio&c  Fluid  

Newborn   Teen   30   50   80  

Pluripotency   Prolifera0on   Gene0c  Stability  

Comments  

Embryonic   To#potent   Very  High   Not  stable   Ethical  considera#on  

Fetal   Pluripotent   High   Stable   Easily  collected  from  amnio0c  fluid,  placenta,  etc.  

Adult   Mul#potent  or  Unipotent  

Low   Stable   O?en,  hard  to  harvest  (bone  marrow,  fat,  muscle)  

Induced  Pluripotent  

Pluripotent   Varied    

Varied   Reverse  engineered  from  adult  #ssue  

(A)DiaphragmaticTendon[Fuchs 2004]

(B)TracheaConstruct[Kunisaki 2006]

(C)BoneGraft[Steigman 2009]

(continue on reverse)

Experimental Methods•IRBApprovalStElizabeth’sHospitalandChildren’sHospitalinBoston(2008-present)

•IRBapprovalStElizabeth’sHospital&BiocellCenter(2010-present)•Allpatientcounseledfirstforamniocentesisforvariousobstetricalindications:

•Fetal Lung Maturity (FLM) amniocentesis•Late Genetic amniocentesis (defined as >20 weeks gestation)•Paternity testing•Twin-to-Twin Transfusion Syndrome and other polyhydramnios

Figure 2. Experimental Workflow

Results and Discussion•78+%ofsamplesfrom3dtrimesterinBiocellCenterstudy,66%ofsamplesfromBostonChildren’sand60%inTOMAstudiesdemonstratedviablecellgrowthinculture(comparedto~95%ofsamplescollectedduring2ndtrimester[TOMA, Biocell Center data])(Table1)

•Thissomewhatlowerpercentage,eventhoughstillimpressivelyhigh,mightbeduetopresenceofothercomponentsintheamnioticfluidatlategestationthatcanaffectviabilityofstemcell(i.e.higherconcentrationoffetallungsurfactant)

•Itisworthnotingthatsuccessrateofstemcellscollectionsfromamnioticfluidcomparesfavorablytootherstemcellcollectionmodalities.Forexample,collectionofumbilicalcordbloodresultsin~50%ofallsamplesproducingsufficientquantitiesofhematopoieticstemcells[National Marrow Donor Program data]

Table 1. Third Trimester Amniotic Fluid Contains Viable MSCs.

•PercentageofsampleswithviableMSCswascomparablebetweenthreegroups,andcanbefurtherimprovedwithbettersamplingandmodifiedculturingtechniques

1 - Total of 60 samples were collected at both 2nd and 3d trimester2 - Some samples for Children’s’ studies were collected using amnio reduction – i.e. 1+ liter of fluid that might have affected the viability studies for MSCs3 – During the initial culture, 7 out of 9 samples showed viable cell growth. During second culture attempt, including modifications to culture protocol, 8 out of 9 (89%) samples produced viable cells, including one of the samples that did not grow during the first culture attempt.4 – Most samples collected just prior to elective C-section.

Results and Discussion (continued)•CellsfromthirdtrimestersampleswereanalyzedbasedonthecriteriaofMSCsdefinedbytheInternationalSocietyofCellularTherapy[Position Paper]:•Fibroblastic morphology•Positive expression of surface markers CD73, CD90 and CD105, and negative expression of surface

markers CD14, CD34, CD45, CD19 and HLA-DR•Differentiation into cells of all three germ layers, such as adipogenic, osteogenic and chondrogenic

lineages•Allculturedsamplesdisplayedcellswithdistinctfibroblasticmorphology,characteristicofMSCs(Figure2,A.)

• TheMSCsphenotypeofcellsin3dtrimesteramnioticfluidwasfurtherconfirmedbyFACSanalysis.AlltestedsampleshadexpressionprofilecharacteristictoMSCs•the positive expression of CD73, CD90 and CD105 surface

molecules•negative expression of hematopoietic markers as CD14, CD34,

CD45, CD19 and HLA-DR •One sample in Biocell Center study had less than 10% of cells

expressing CD90 and 40% of cells e xpressing CD105. Further analysis of this sample is on-going

• Severalsamplesweretestedindifferentiationexperimentsanddemonstrateddifferentiationintoadipogenic,chondrogenicandosteogeniclineages(Figure2,B.)Completedifferentiationanalysisofallcollectedsamplesison-going

Figure 3. Cells from Third Trimester Amniotic Fluid Meet the Defining

Criteria of MSCs (A) Cells cultured from third trimester amniotic fluid have fibroblastic morphology characteristic to MSCs (Biocell Center data)

(B) Third trimester amniotic fluid MSCs can differentiate in cells of adipogenic, osteogenic and chondrogenic lineages (TOMA data)

Conclusions•Itispossibletoisolate,cryopreserveandexpandAF-MSCatlatestagesofgestation.TheyarecomparabletoyoungerAF-MSCscollectedduringsecondtrimester.

•Thirdtrimestersamplesresultinviablestemcellsis78+%,noticeablyhigherthanmethodsofstemcellscollectionsfromothersources(althoughlowerthan~95+%successrateofMSCscollectionfrom2ndtrimesteramnioticfluid.)

•Thisstudyisongoingtocomparesamplingandcellculturetechniquesinordertofurtherimprovethesuccessrateof3dtrimestercollections.

•WeconcludethatthirdtrimesteramnioticfluidcanserveasanovelsourceofhumanMSCsforclinicalresearchandfamilybanking.

References#  Samples  Collected  

Mean  GA   %  Samples  with  viable  MSCs  

St  E.  /  Boston  Children’s   44  1   34  weeks   66%  2  St.  E.  /  Biocell  Center   9   34.5  weeks   78+%  3  TOMA   17   38.5  weeks   60%  4  

Collect  20-­‐30  cc  during    amnio  for  FLM  or  poly/gene;c  

Process  and  cryopreserve  in  liquid  nitrogen  

Thaw  cells  and  culture  in  MSCs  media  

Confirm  MSC  phenotype  &  viability  

St.  Elizabeth  Hospital  /                                    Biocell  Center  Workflow  

St.  Elizabeth  Hospital  /                                                              Boston  Children’s  Hospital  Workflow  

Collect  10  to  1500  cc    during    amnio  for  FLM  or  poly/gene:c  

Processed  &  spun  down  

Confirm  MSC  phenotype  &  viability  

Remove  pellets,    resuspend  in  DMEM;  then  culture  

St.  Elizabeth  Hospital  /                                                              Boston  Children’s  Hospital  Workflow  

Collect  10  to  1500  cc    during    amnio  for  FLM  or  poly/gene:c  

Processed  &  spun  down  

Confirm  MSC  phenotype  &  viability  

Remove  pellets,    resuspend  in  DMEM;  then  culture  

A.

ADIPO   OSTEO   CHONDRO  B.

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