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Controlling Culture Dynamics for the Expansion of Hematopoietic Stem Cells

Article · Literature Review  · September 2001   with   70 Reads DOI: 10.1089/15258160152509091 · Source: PubMedAbstractThe ex vivo expansion of hematopoietic stem cells (HSCs) is the subject of intense commercial and academic interest due to the potential of HSCs to be a renewable source of material for cellular therapeutics. Unfortunately, because methodologies have not yet been developed to grow clinically relevant numbers of HSCs (or their derivatives) consistently, the potential of this technology is limited. Manipulation of the in vitro culture microenvironment, primarily through cytokine supplementation, has been the predominant approach in studies attempting to expand primary human HSC numbers in vitro. While promising results have been obtained, it is becoming clear that novel methods must be developed before cellular therapies using these stem cells can become routine. Ideally, bioprocesses must be designed to target specifically the growth of stem cell populations while incorporating positive and negative feedback from potentially dynamic mature and maturing cell populations. The product of these culture systems should consist of not only HSCs, but also of cells that allow the engraftment of HSCs and, ideally, cells responsible for the immediate or accelerated functional support of patients. Development of such "designer transplants" will require combining optimal culture conditions capable of amplifying HSC numbers with novel approaches for finely controlling the number, functional capabilities, and characteristics of potentially therapeutic cells in these very complex cell culture systems.

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Join for free Figures JOURNAL OF HEMATOTHERAPY & STE M CELL RESEARCH 1 0:48 1 – 492 ( 200 1)Mar y A nn Lieb ert, Inc .Cutting Edge Comm un icationContro llin g Culture Dynamics for the Expansio n ofHematopoietic S te m CellsG.J . MA D LAM BA YA N ,1, 2I. R OG ERS ,4R. F . C A SPE R,4an d P. W. Z AN DST RA1– 3AB S TRA CTThe ex v ivo ex pan si o n o f hem ato poie tic stem cells (H SCs) is the su bject o f inten se c om merc ial an daca d emic interest d ue to the po tentia l of H SC s to be a renew a ble so urce of m ater i a l f or cellula rther a peutics. Un fortu nate ly, beca use m etho dolo gies h av e no t y et b een d evelo ped to g row c li n icallyrelev a nt n umb ers of HSC s (or the ir der i v ativ es) co nsistently, th e po tentia l of this techn olog y is lim -ited. M anipu l a tion o f the in v itro cultu re micro enviro n men t, prim arily thr oug h c yto kine supp le-me nta tion , ha s been the pr edom ina nt ap proa ch in stud i e s a ttem pting to ex pan d p rima ry hu ma nHSC nu mbe rs in v itro. W hile p rom ising r esults ha v e be en o btain ed, it is beco ming c lear t hat no velme tho ds m ust be dev elop ed befo re cellula r thera pies using the se stem ce l ls ca n b ecom e ro utine. Id e-ally, bio pro cesses mu st be d esigned to ta rget specifica lly the gr o wth o f ste m cell po pula tions whileinco rpo ra ting p ositive and nega tive fee dba ck fro m pote ntially d yna mic m atu re a nd ma turing ce l lpo pu l atio ns. T he pr odu ct of th ese cu l t ure system s sho uld co nsist of n ot o nly HS Cs, b ut a ls o o f cellstha t a l lo w the eng raf tme nt of HS Cs a nd, id eally, cells respo nsible fo r t he im me diate o r a ccelera tedfunc tio nal sup port of pa tients. D ev elopm ent of such “ designe r tr anspla nts” w i ll requ ire co mb iningop tim al cu lture con ditions ca pab le o f am plify ing HS C n um bers w i t h no vel ap pro ache s for fine lycon tr olling t he nu mbe r, fun ction al ca pab ilities, and cha ract eristics of po tent i a lly thera peu tic cellsin these v e ry co m plex c ell cu l t ure sy stems.48 1OV ER VIE WIN TE R E ST IN H E MA T O PO IE TI C S TE M C EL LS (HSCs ) h as gro wn o ver t he pas t dec ade b ecau se o f th eir p ote nti alto p rovi de essen tial sta rtin g m ateri al fo r ne xt-g ene ratio nthe rapeu t ics, i ncl udi ng gen e, cel lul ar, and tissu e re gen -erati on the rapie s. Alth ou gh the first t wo u ses h ave bee nthe su bje ct of i nte nse in vest iga tio n, the latt er i s o ne o fthe fast est g rowi ng a reas i n st em ce ll researc h. T he p o-ten tial of u mbi lica l cord b loo d (UC B)-d erive d HS Cs inth e co nt ext of ti ssue en gin eerin g an d ce ll trans plan tat ionhas b een fu rthe r en ha nced b y the recen t d emon strat ionth at th is cell sourc e may be mo re i mmu nol og ical ly naïv eth an ad ult - d eriv ed HSCs (1 –4 ). T his fin di ng, a lon g wi thth e o bserv ati on tha t hi gh HSC d os e the rapi es can resu ltin sta b le ch ime ric h emat opo iesi s (5 ,6), m ay p rov ide r o -bu st s trate gies fo r allo ge nei c orga n o r ti ssu e tran spl ant a-tio n. R ecen t find ing s, i n whi ch b on e marrow-d eriv edcel ls hav e bee n sh own to di fferent iat e i nto n on -blo od tis-sue s (7–1 0), h a ve b road ene d th e p oten tia l use o f HSC s1Ins ti tute o f Bi omat eria ls a nd Bio medi cal En gin e erin g, 2Dep ar tmen ts of Ch emic al En gin eer ing an d App lied Ch emis try a nd3Anato my and Cell Bio log y, Un ive rsity o f To ron to a nd 4Samu el Lune nf eld Re sea rch Inst itut e, Mo unt Si nai Ho spi tal, Toro nto ,Ont ar io, Ca nad a. and c hal len ged the d og ma tha t t he fa te o f stem cell s i srestri cted to thei r t issu e(s) or o rigi n. Alth ou gh thes e stu d-ies hav e no t, in g ene ral, rig oro usly diffe renti ate d be tweenHSCs a nd o th er c ell ty pes kn own to exi st in t he bo nemarrow (11 ), so me hav e c lea r l y in di cate d t hat cell s wi ththe ch aract erist ics o f HSCs m ay be the acti ve p roge ni-tors in t his p h eno men on (9, 12). Th u s, p rov idi ng a n ex-pan dab l e so urce o f HSC s m ay n ot o nly en abl e he mato -po ieti c c ell an d g ene th erap y ap pli cati on s, bu t now h asthe a dde d p ote nti al of pro vid ing c ell s for t iss ue re gen er-ati on th erapi es.The pro mise o f HSC s fo r cl ini cal th erap y , h owev er, i slim ited bec ause t he nu mber of st em ce lls th at can be ob -tai ned fro m t radi tio nal sou rces i n clud in g UCB, b on e m ar-row (BM ), an d mo bili zed p erip hera l blo od (PB) i s in ad-equ ate . Th erefore , effec tiv e, te chn olo gi call y rel eva ntmeth od o log ies to c ult iva te th ese c ells in v itro ha ve be endiffi cul t t o d evi se. For ex amp le, a lth ou gh UC B repre sent sa rich sou r c e of tran spl ant abl e H SCs (1 3–16) tha t can beeasi ly ob tain ed fro m ti ssue no rmall y di scard ed at b irth;cel ls ob tain ed fro m a sin gle co rd bl ood col lect ion , m oreofte n t han n ot, cont ai n to o few HSC s to ensu re t he hem a-tol og ic al en graft ment of an adu lt p ati ent (17 ,18 ). In o urexp erie nc e, 7 5% of th e gre ater tha n 4 00 0 b ank ed sa m-ple s at th e To ront o Umbi lic al Cord Bl ood B ank c on tainon ly e n oug h HSCs to b e u seful fo r p edi atric b on e m ar-row tra nspl ant ati ons (1 9). For the se ba nke d sam ple s tobe u seful fo r si ngl e o r mu lti ple a dul t t r a nsp lan ts, or fo rmul tip le ti ssue regen e ratio n the rapie s, t hei r co nte nt o fHSCs mu st be exp and ed.IN VIV O A ND IN V ITR O M EAS UR EM EN TSOF HSC EX PA NSIO NAll st em c ells, rega rdle ss of t hei r tissu e o f orig in, a recel ls tha t ha ve an e xten siv e ca pac ity for self-ren ewal an dmain tai n th e cap abi lity to d ifferen tiat e i nto m atu re, fun c-tio nal ce lls. Ti ll an d M cCu llo ch (20 ) pro vi ded th e firstexp erim en tal ev ide nc e sug ges tin g t he e xist enc e of su chcel ls in th e hem ato poi etic s yste m whe n t hey ob serve d t heformat ion of seria lly tran spl an tab le m ult ili neag e c olo nie sderi ved from s ing le cell s in the sple en of l eth ally irrad i-ate d mi ce. Su bseq ue nt ex peri ment s demo nst ratin g thatlon g-t erm eng ra f tme n t of bo th th e l ymp hoi d an d m yel o idlin eag es c an b e ac hie ved by the pro gen y of a sin gl emuri ne (2 1–2 3) or hu man (2 4) cel l, and th at th e pro gen yof a si ngl e c lon e ca n re pop ul ate m ult ipl e seco nd ary re-cip ien ts (2 5,2 6), h as sh own t hat so me o f t hes e very p rim-iti ve cell s are c apab le of ex tens ive (.100 0-fo ld ) ( 2 7) inviv o amp lific at ion .Armed wi th t his k no wled ge, a si g nific ant e ffort ha sbee n di recte d at try ing to re capi t ula te t his in viv o ex -pan sio n ca paci ty i n vi tro. Hu man h emat op oie tic c ell c ul-ture s are typi ca lly in iti ated wi th c ells p he not ypi cal lycha racte riz ed as exp ressin g th e ce ll-su rf a ce ant ig en CD3 4(28 ) an d p erha ps lack in g th e C D38 ant ige n, i.e.,CD3 41CD382cel ls (29 ). C ell s i sola ted base d on ot hermark ers, s uch a s th e ex pressi on o f Th y-1 or AC 133(30 – 3 3), o r ev en t he l ack of C D34 or HLA-DR ex pres-sio n (22, 3 4,3 5), hav e also be en sh own to d isp lay l ong -term h emat opo iet ic p ot ent ial. T hese p hen ot ypi c ma rkersare us eful n ot o nly fo r the i nit ial i sola tio n o f ce lls th atare enri ched for HSCs (thu s al lowi ng th e i nit iat ion of c ul-ture s with hi g her d ensi ties o f su ch ce lls), bu t a lso b ecau sethe y h ave the p oten tia l to all ow t he rap id , rela tiv ely in-exp ens iv e det erm inat ion o f cu ltu re s tatu s. In add i tio n tothe se an aly tica l a dv ant ages , ph eno typ ic ch arac teriz atio nof HSC s in p arti cula rly ap pea lin g bec au se it pro vid e s th eon ly ph ysic al app roac h av ail abl e t o ma nip ula te t he nu m-bers an d t ype s o f cel ls g en erate d du ring in v itro c ult ure(the co mpo siti on o f t he cu ltu res can b e i ndi rectl y m a-nip ul at ed, for ex amp le b y su pp l emen tati on wi th spe cifi ccyt ok in es; s ee be low). Un fort una tel y, mu ch o f wha t iskn own ab ou t re lati on ship s b etwe en t he ph en oty pe o fHSCs, a nd th eir fun cti on o r g eno ty pe (36 ,37 ) h as be endet ermin e d by flo w c yto met r i c so r t ing o f u ncu lt uredcel ls; an in creasi ng b od y of e vid enc e su gg ests th at the invit ro cul ture en v iron men t may rapid ly d isso ciat e est ab -lish ed fun cti ona l an d ph en oty pic rel atio nsh ip s (38 ,3 9).Ultim atel y , pres ervat ion of ce ll fun cti on i s t he d eter-min ing c riteri a i n the succ ess of in v itro hu man HSC c ul-ture syst ems. Cel l f u nc tio n can b e que ried in v itro u singthe co lon y-fo rming c ell (CFC ) a nd l on g-term cu ltu re-in i-tia tin g cell (LTC - IC ) assa ys, tec hn iqu es th at de te ct t h eprese nce o f comm itt ed an d m ult ip ote nt c ells o n th e b a-sis of the fo rmat ion o f mo rpho lo gic al ly d istin g ui shab lecol on ie s eit her d irect ly in m ethy lce llu lose c ult ures(CFC ), o r afte r grea ter t han 5 week s o n stro mal feed erlay ers (LTC-IC ) (40–4 2). In vi vo func tio nal ass ays fo rHSCs ha v e be en d eve lo ped o n th e b asis o f th e abi lity o fthe se cell s t o recons titu t e h ema top oie sis in th e BM of s e-vere comb in ed immu no defic ien t (SC ID) an d no no besedia bet ic (NOD) mi ce fo llo wing in trav eno us i nje ctio n(43 – 4 5). T hese so -call ed NO D/SCID-rep op u lati ng ce lls(SCID rep op ula tin g c ell s) (46) a re con sid ered hu manHSCs t hat h ome to an d eng raft th e mu rine bo n e ma rrow,where t hey su bseq uen t ly pro life r a te a nd d ifferen tia te i ntomul tip le lin eag es (47 –49).Impo rtant l y, p hen oty pi c a nal ysi s, i n v itro fun ctio nalassay s, an d i n vi vo xe no gen ic as says o f hu man h ema to-po ieti c st em ce ll p ote nti al a re al l surrog ate me asure s o fhu man HSC p ote nti al and , as su ch, e ach q uery t he t estcel l p op ula tio n for p arti cul ar p rop ertie s. W het her it is th eexp ressi on of c ell-s urface an tig ens , th e a bil ity to ma in-tai n hema top oi esis i n vit ro for e x ten ded peri ods of ti me,or t he c apac ity t o h ome to a nd di fferent iat e i n mo use bo nemarrow , each o n it s o wn p r o vi des a sp ecifi c a naly tic alins igh t in to the pro pert ies o f hu man HSC s. Alth oug htak en tog eth er thes e assa ys offer a co mpre he nsi ve p ic-MADL AMB AYAN ET AL .482 ture of hu man HSCs , it i s no tewo rth y th at the y do no t al-ways det ect the s ame po pu la tio n o f cel ls. P revio u s stu d-ies h ave d emo nstra ted that SC ID rep op ula tin g cell s a rebio lo gi cal ly d isti n ct fro m at lea st some LT C-ICs. For ex-amp le, Laro che lle et al . (5 0) o bse rved tha t LTC -ICs an dCFC s a re inc apab le of en graft ing NOD/SC ID mic e andare le ss lik ely to b e t ransd uce d us ing retrov iral ge n e t rans-fer. Th e con verse h old s t rue for LTC - IC s an d CF Cs.Othe r inv esti gat ors ha ve sug gest ed tha t SCID rep op ula t-ing c ells oc cur a t l ower freq ue ncie s th an LTC -ICs i n B M,PB, and UCB , sug gest ing th at LTC -ICs rep resen t a h e t-erog ene o us p op ulat ion made u p o f b ot h p r i mit ive an dmore restri cted pro gen i tors (16 ,42 ). Th e ex tent of co rre-lat ion bet ween SC ID repo pul ati ng c ells a nd L TC-ICs i slik ely de pen den t u po n th e me tho do log y us ed to ass ayeach pa r t icu lar ou tpu t; SC ID rep op ulat ing c ell m easu re-men t i s dep end ent upo n in jec tio n d ose an d time o f dif-feren tiat ed cel l d etec tio n (48 ,49 ,5 1,5 2), wh ile LTC-ICdet ecti o n is de pen den t u pon th e t ime o f cul ture on sup -po rtiv e strom al ce lls, as we ll as t he i den ti ty o f th e stro -mal c ell p op ula ti on (4 0,5 3). In each of th ese assay s, i t i scle ar t hat ex ten d ed de tect ion ti me p oi nts rea d ou t in-creas ing ly p rimit ive ce ll pop ula tio ns (47 ,5 3,5 4), an d thatat leas t so me co nd itio ns ex ist whe re the se a ssay s de tectov erlap p ing cel l po pul ati ons (5 5–5 7). Si mila r c omp lic a-tio ns ex ist wh en co mpa ring func tio nal an d ph eno typ icou tpu ts . For ex amp le, al tho ugh un cu ltu r e d SCID re pop -ula tin g c ells hav e b een exc lusi vel y a ssoci ated with t heCD3 41CD382ph eno t ype (5 8), it is cle ar t hat some cu l-ture con di ti ons whic h resu lt in a n in creas e ofCD3 41CD382cell s d o no t o utp ut prop ort ion al in cre asesin SC I D repo pul ati ng cel l n umb ers (59 ,60 ). Fin all y, a sall ude d t o earli er, i t ha s be en sho wn th at re pop u lat ingcel ls ma y a lso be pre sent i n t he CD342pop ul at ion(61 ,62 ), a nd t hat t he C D34 an tig en may i tsel f b e reg u-lat ed i nde pen den tly o f HSC p ote nti al (63 ,64 ).Th is b rie f o ve rv ie w of t he r el ati o n sh i p s b et we en th edi ffe re nt a ssa y s u se d t o m ea sur e H SC a ct iv i ty an d th eab se n ce o f ri g oro u s st an d ard s in t he u se o f t h e se as sa ysca ut i on s a ga in st t he u se of an y o n e o f t he se te ch ni q u esto o p ti mi z e cu l tu re c on d it io ns fo r th e e xp an si o n o ftra ns p la nt able HSC s. Ult im a te ly , cl in i ca ll y t ran s-pl an t ab l e H SCs wi ll ha ve t o re ta in l on g -te rm rep o pu -la ti n g ab il it y in h um an s; m o vi ng t ow ard t hi s e nd p oi ntwil l re q ui re in t er rog at ion o f H SC p rop e rti es fro m m ul -ti pl e pe rsp ec ti ve s.Th e in vi tro se lf -ren ew al o f h um an HSC s h as b ee nco nv in ci n g l y d em on st rat ed u si n g p h en ot yp ic ass ay s,an d co nfi rm ed b y b ot h in v it ro a nd in v iv o f un ct io na las say s . To d at e, ho we ve r, on l y a m od e st a nd t ran si en tex pa n si on o f c el ls c ap ab le of re po p ul at in g N OD/ SC IDmi ce h as be en c on si ste n tl y a ch i ev ed . C o nn ea ll y et al .(5 5) sh ow ed th a t CD3 41CD 382UC B ce ll s gro wn fo r5–8 d a ys i n se rum -fre e ex p a ns io n c ul tu re s c ou ld p ro -du ce a t wo- fol d i nc rea se in SR Cs , wh er eas B h ati a et a l.(5 8) an d Ued a e t a l. (6 5) bo t h rep o rte d a sl ig ht ly h i gh erfo ld e x pa ns io n (,fo ur-f o ld ). It is no te wo rth y th a t a fe wst ud i es , p rim a ril y ba sed o n sm al l-s ca le a dh ere nt (s tro -ma l-b a se d) c ul tu re s, h av e re po rt ed su st ai ne d i n v it roHSC e xp an si on (66 –6 8); fo r ex am pl e, Pi ac ib el lo e t a l.de sc ri be d c al cu l at ed SR C ex p an sio n s o f a pp ro x ima te l y70 -fo l d o ve r a 9 - t o 1 0 -wee k pe rio d . A lt ho u gh en co u r-ag in g , t he se s ys te ms a r e no t y et ap p li c ab le to c li n ica lex pa n si on sy st ems , e it h er d ue t o t h eir t ran si en t n at u reor b ec au se o f th ei r d ep en d en ce o n l ow ce ll d en si ti es ,st rom a l cel ls, o r ex te n si ve c ul t ur e ti me . W ha t is no w re-qu ir e d i s t h e i de nt if ica ti o n o f a c li n ic all y re le va n t c ul -tu re sys t em c ap ab l e o f a ch ie vi ng s us ta i ne d HSC g ro wth .MA N IPU LA TION OF IN V ITRO CU LTU R EDY N AM IC S US I N G P RO TEINMO D ULA TO RS O F C ELL F UN CT IONHSCs must main tain a b ala nce b etwe en self-ren ewin gcel l di visi ons (need ed to en sure t heir e xis tenc e th roug h-ou t th e l ife of an o rgan ism) a nd di fferent ia tio n (n eed edto re plac e t h e l oss of m ature ce ll pop ula tio ns). In vi vo ,the av erag e t ime nee ded fo r an ad ult HSC to en ter t hecel l c ycl e v arie s dep e ndi ng on t he o rga nism . Ad ultmuri ne HSC s en ter int o t he ce ll cy cle onc e eve ry 30 – 5 7day s (6 9,7 0), whe reas muc h lon ger ti me pe riod s were ob -serve d in no n-h um an pri mate s (bab oo ns) where o nl y 50 %of HSC s d ivi ded ov er a 12 -mon th perio d (71 ).Th e su cc ess ful m ai nt en a nc e a nd ex p an si on o f tra n s-pl an t ab l e HSC s in vi tro re qu i res t rig g eri ng st em ce ll s topr ol i fera te wi th o ut un d erg o in g di ffe ren ti at i o n . C u lt ure dHSC s m us t ad d it io na l l y a vo id a po p t osi s a n d m ai nt ai nth e ab il it y t o h om e, e ng raft , a n d d iffe re nt ia te in to a p-pr op ri at e re ce pt iv e t is su es. B ec au se t he re gu l at io n ofth es e pro c ess es i nv ol v es in t era ct io n s b et wee n ce ll -su r-fac e r ec ep to rs an d a la rge g ro u p of s ol ub le an d m at rix -bo u n d m od u la to rs o f c el l fu n ct io n, ma ny i nv es ti ga to r sha ve fo cu se d on de v el op in g c ul tu re sy st em s su p pl e-me nt e d wi th a pp ro pr ia te c om bi na ti o n s of st imu l at oryfac to r s t o m ai nt ai n an d ex pa nd HSC s i n v it ro . Th e c y-to ki n e m ic roe nv i ronme nt i n t h e se c u lt ure s is dy n ami c(7 2), wi th m ul ti pl e ce ll t yp e s c om p eti n g fo r cy to k in esan d nu tr ien t s, ma n y of w hi ch ar e ca pa bl e o f i n flu en c-in g s te m ce l l f at e d ir ec tl y o r i nd ire ct ly . T he se c om pl exin te ra ct io n s m ak e t h e cy t ok in e c o mp os it io n of HS C ex -pa ns i on me d iu m p a rti cu la rly ch al l en g in g t o o pt im iz e.Fu rth e rmo re , as c ul tu re t ime p ro gr ess es, th e c o mp o si-ti on o f th e c ul tu re sy st em ca n c h an g e d ram at i ca ll y a ndth e e n do ge no u s p ro du ct io n o f fa c to rs th at h av e a s up -pr ess i ng e ffec t o n H SC s el f-re ne wal ma y si g ni fic an tl yin fl u en ce c ul tu re o ut pu t. D esi g n in g s uc ce ssfu l HSC e x-pa ns i on cu l tu res th u s re qu ire s st ra te gi es to ma in ta in a nap pr o pri at e b ala n ce b e twe en st im ul at ory a n d in hi b it orymo du lat o rs of H SC fun ct io n .HSC EXPANSION483 Po sitive mo du l a tors of H SC fun ctionFacto rial ( 3 8) an d co mpo site d esig n met ho ds h av ebee n u sed t o in crea se the effici enc y of i nv estig ati on s o fthe effect s o f sti mul ato ry fact ors o n HSCs an d to reve alun exp ec ted i nte racti on s that wo uld oth erwise be miss edby co nv ent ion al do se–resp ons e a nal yses. Th ese op ti-miza tio n me tho ds a re adv an tag eou s b ecau se t hey can beapp lie d to co mple x systems in wh ich mec han isti c d eta ilsare no t fu lly u nde rstoo d. Exa mpl es o f w here thi s ap -pro ach h as be en succ essful inc lud e: (1) t he id en tific atio nof p artic ular fa cto r s th at i nflu enc e HSC self-re newal[e.g ., fl t-3/flk -2 l iga nd (7 3)], (2 ) th e d emo nst r a tio n thatthe c yto kin e re qui remen ts o f HSCs ma y ch an ge d urin ghu man o nto ge ny (7 4), an d (3) the ob serv atio n of a th resh-old cy tok ine co nce ntra tio n effec t fo r HSC se lf-rene waland di f fe rent iati on ( 38 ,75 ).Amon g the differe nt stim ula tory cy tok in es i den tifi edin t he a bo ve men tio ne d an d ot her st udi es, co mbi nat ion sof stem c ell fa cto r (SCF ) , flt -3/flk-2 l igan d (FL ) , an dthro mbo p oie tin (TPO) h av e b een sh own to b e pa rticu -larl y e ffecti ve i n el icit ing se lf-rene wal di visi ons o f hu -man HSC s. SCF , t he l iga nd for t he t yrosi ne k ina se re-cep tor c -Kit, p romo tes th e su rviv al an d g rowth ofprim itiv e h emat op oie tic prog eni tors wh en u sed in c om-bin ati o n wit h o the r sti mul ato ry c y tok ine s (76, 77); li ttl eexp ans io n i s obse rved i n c ult ures su ppl emen ted wit h SC Falo ne (78 ). FL, a fa ctor sec reted by stro mal cel ls an dwhic h ha s doc ume nte d si mila riti es t o SCF, h as al so be enext ensi v ely i nv esti gate d as a po ten tial me dia tor of HSCexp ans io n and m aint ena nce (79 ). FL i s b eli eved to ac t b yrecru itin g HSCs int o th e cell cy cle (80 ,81 ) an d b y pro -mot ing surv iva l, pre suma bly t hrou gh i nh ibi tio n of a pop -tos is (8 2–84 ). T PO su pp orts t he prol iferat ion and l ong -term m ain ten ance of pri miti ve h emat op oie tic pro gen ito rsand rep opu lat ing HSC s wh en u sed i n c omb ina tio n wi thoth er stimu lat ory c yto ki nes (8 5–89 ). Wh en use d alo ne ,TPO h as litt le e f fe ct o n the ex pa nsio n o f HSC s i n su s-pen sio n cul ture s (90 ,91 ). Ho weve r, in th e p resen ce ofstrom al c ells , T PO m ay p ro mote sust ain ed HSC exp an-sio n (68). T hus, a s p ost ula ted by B artel mez and c ol-lea gue s (6 8), T PO ma y p lay a ke y ro le i n th e lo ng -termmain ten a nce an d de vel opm ent o f p rimi tiv e HSC s in vi vo ,and m ay be a usefu l add iti ve for su cces sful HS C ex pan -sio n e x v ivo . Alt hou gh mo st stu di es h av e fo cuse d on “ he-mato po i etic ” cyt oki nes, o the r g rou p s h ave i den tifi edno vel fac tors, no t ori gin all y id en tifie d as a resul t of th eireffect s on h ema top oie tic c ells , th at m ay regu lat e HSC re-spo nses . Fo r ex amp le, B hat ia e t a l. (92) repo rted th atbo ne morp hog en etic pro tei ns (BM Ps) c an m odu l ate t hepro liferat i on a nd d ifferen tia tio n of p rimit ive hu man h e-mato po i etic cel ls (C D341CD3 82lin2) a nd t hat B MPtyp e-1 re cept ors were exp ressed , a lon g w ith d own strea mtran sdu ce rs of t he BM P si gna lli ng pa thway , in sub pop u-lat ion s of h emat op oie tic pro gen ito rs. Rece ntl y, th e a dd i-tio n o f the Not ch l iga nd, Jag ged -1, to cul tures o f p rimi-tiv e pro gen ito r cel ls in du ced the su r v iv al an d e xpa nsio nof rep opu lat ing HSCs (93 ) im pli cati ng Ja gg ed-1 a s an -oth er no ve l gro wth fac tor for t he mo dul atio n of hum anHSC fu n ctio n (9 4).Neg a t ive mo du lators of H SC fu nctionIn sp ite o f ex ten siv e in vest iga tio ns, i n v itro e xpa nsio nsyst ems sup plem en ted wit h co mbi nat ion s o f st imu lato rycyt ok in es h ave no t, to date , y ield ed su stai ned HSCgro wth, sug gest ing tha t HSC ex pan sion may b e l imit edby ot her fa ctors . In vi tro p rod uct ion o f in hib ito ry p r o tei nsis o ne p ote nti al re aso n for th e tran sien t e xpa n sion o b-serve d i n man y c ult ures. Pro tei n facto rs, su ch as tran s-formi ng grow th f a cto r-b(TGF-b), tumo r n ecros is facto r-a(TNF-a), an d, more rec ent ly, in terl euk in-3 (IL-3 ) h av ebee n s hown t o d ecrea se repo pu l atin g po ten tia l b y ind uc-ing d ifferen tia tio n or ap op tosi s, as we ll as by d ecrea singthe a bil ity of rep op ula tin g st em cel ls to mig rate to t hebo ne m arrow mi croe nvi ronm ent (see b elo w). Oth er fac-tors kno wn t o e l icit inh ib it ory resp ons es i ncl ude macro -ph age infl amma tory p rote in-1a(MIP-1a) (95, 96 ) andmon ocy t e c hemo att racta nt p rotei n-1 (M CP-1 ) (97 ). Fo rthe se i nhi bit ory cyt ok in es t o a ffect HSC g rowt h in v itro ,the y must b e gen erate d i n cul ture. Ev ide nce s howi ng th atthi s occ urs h as be en obt ain ed from t he an aly sis of h e-mato po i etic p rog enit or c ult ures, wh ere b ot h mRNA andpro tein e xpre ssio n o f inh ibi tory a nd stimu lat ory fac torshav e bee n m easu red fro m st romal and p rog eni tor ce llpo pul at ion s (9 8,9 9).Ideal ly, th e goa l o f in v itro HSC cu ltu re sy st ems is torecap itu l ate the relat ive ly h igh rat e of HS C se lf-rene waland pro life r a tio n ob serve d d urin g e mbry oge nesi s (1 00 ).Rel ati on shi ps b etwee n th e e ffects of c yto kin es an dgro wth fac tors an d th eir i mpa ct on th e rate of ce ll c yclepro gressi o n a nd t h e i nit iat ion of n ew g ene e xp r e ssio n pro -file s r e qu ired fo r hema top oi etic di fferent iat ion h av e b eeninv est ig ate d i n se vera l d eve lop men tal mo de ls (10 1–10 4).For ex amp le, d urin g l i mb bu d d evel op ment , dire ctl y a l-teri ng th e ra te o f cell cyc le p rogre ssion c an resu lt in gen eexp ressi on ch ang es asso ciat ed with dis tin ct func tio ns o fdiffe renti a ted c ell s (10 5). C ell s ca n respo nd t o ex og eno uscyt ok in es, bot h s timu lat ory an d in hib ito ry, by way o f adis tin ct set o f seri ne / t h reon ine kin ases, ca lled c ycli n d e-pen den t k ina se(s), or cd k(s), du e t o t hei r as soci atio n wi thsho rt-liv e d reg ula tory prot ein s c alle d cy cli ns (10 6). Es-tab lish i ng con nec tio ns betw een p roli ferati on an d differ-ent iat io n, b y und ersta nd ing th e effects of stim u lato ry an dinh ib it ory cy to kin es o n the ra te o f cell cy cle pro gressi on ,may p rove to b e a p owe rf u l t ool fo r ste m c ell main te-nan ce a nd ex pan si on.A spec ific ex amp le of t his ce ll cy cle -relat ed mod ul a-tio n o f ce ll fun cti on re leva nt t o h emat op oie sis c an befou nd i n th e mech a nism o f ac tio n of T GF-b, a cyto ki neMADL AMB AYAN ET AL .484 tha t is se creted by B M stroma l cell s (9 7,1 07 ,10 8), a s wellas ot her d ifferen tia ted cel l p op ula tio ns (9 8), an d kn ownfor it s an tip roli ferati ve effect s o n HSC s (8 6,1 09– 1 14 ).TGF-b’s mai n funct io n i s the inh ibi tio n of t he sh ift fro mG0/G1to S p hase by th e up -regu lat ion o f th e cd k i nh ib-ito rs p 15 , p 27, an d p 21 (1 15 ) . Th e ab ili ty o f qu i esce nthem ato po iet ic ste m c ells t o p rod uce TG F-bin an au -toc rine m ann er pro bab ly a ccou nt s fo r t hei r c ont inu ed q ui-esce nce i n vi vo (1 12). T his i s furt her su p port ed b y th eob serva ti on th at th e add itio n o f n eutra lizi ng an ti-TG F-bant ibo d ies (11 6,1 17 ), or o f an tise nse o lig ome rs t o the D2cyc lin in hi bit or p2 7ki p 1(11 8), allo ws fo r the prol iferat io nand ma int ena nce o f HSCs i n v itro . The ac tio n of TGF-bon cy cli ng ce lls, or in th e pre senc e of st imu lat ory fa c-tors, is mo r e co mpli cat ed (1 15). T GF-bha s be en sh ownto len gth en t he G1pha se of th e cel l cyc le, po ssib ly a l-lowi ng for chro mati n rec on f i gu r a tio n and t he in du ctio nof d ifferen tia tion ; SC F an d FL ma y prev ent HSC di ffer-ent iat io n, at lea st i n p art, b y s peci fical ly s hort eni ng t heG1pha se o f t he ce ll c ycle (119 ,1 20 ). T he obse rva tio n th atTGF-bmay elic it so me of i ts ac tio ns by d own - re gul at-ing t he ex pressi on o f many re cep tor t ype s (1 15 ) , inc lud -ing th ose wh ose si gn alin g ha s be en sh own to b e im por-tan t fo r the in vi tro gro wth and ex pan sio n of HSC , e .g. ,SCF (121 ) an d TPO (86 ), sug gest s tha t a b ala nce b etwe enstim ula to ry and inh ib ito r y fact ors, a nd t hei r effect o n t hecel l cycl e, m ay be re qui red for su stai ned HSC p rolife ra-tio n.Othe r mec han isms al so exi st t hat ca n d ecreas e t heov erall e xpa nsio n o f HSCs. For ex amp le, it h as b eensho wn th at th e presen c e of TNF-ain cu ltu res sup pl e-men ted wit h st imu lato ry cyto ki nes inc lud ing SCF (122 )and FL (12 3,1 24) c an po ten tly inh ib it th e prol iferat ion o fpro gen it or ce lls, l ike ly b y p romo tin g a po pto sis (111 )thro ug h Fas (a memb er o f the TNF re cep tor famil y) sig-nal ing (1 25 ) . IL-3 is ano the r cy tok in e th oug ht to h ave in-hib ito ry f un cti o ns. It is a c ont rove rsia l c yto kin e bec aus eof co nfli ctin g reports rega rdin g it s ab ili ty to stim ula te orinh ib it HSC e xpa nsio n. IL-3 h as b een l ink ed to t hegro wth of p rimit ive cells, in clu din g LT C-ICs a nd CFCs ,and is ofte n fo und in c yto ki ne c omb ina tio ns rep orted tobe effecti ve in ex pan sio n cu ltu res (12 6 ) . Co nv ersel y, sev-eral stu die s h ave in di cate d t hat IL-3 ca n ab roga te the ex-pan sio n a nd se lf-rene wal of prim itiv e s tem cell s in a con -cen trati o n-de pen den t ma nne r (3 8) an d, i n bot h hu manand mu rine mo del s, IL-3 ha s b een sh own to im pai r th ereco nsti tu tin g a bil ity of HSC s (65 ,1 27 ,12 8). The se o b -serva tio n s are so mewha t cl arifie d by t he rece nt fin d ingtha t IL-3 may p reve nt HSC s from h omi ng t o th e b o nemarrow by i mpa irin g t hei r ch emo tac tic resp ons e to stro -mal deriv ed fac tor-1 (SDF -1) thro ug h th e CXCR 4 re-cep tor (1 29), th ereb y re sult ing i n the i n viv o clea ranc eand d estru cti on o f p ote nti al en graft ing ce lls in no n-hem ato po iet ic t issue s.Take n t oge the r, t hes e s tud ies su gge st th at inh ibi to ryfacto rs ma y pl ay a ma jor rol e in d ecrea sing b oth th e ac-tua l and m easu red i n vit ro e xpa nsio n o f rep opu lat ingHSCs by ca usin g th em to al ter th eir ce ll cy cle ra te (andpo ssibl y in d uci ng d ifferen tiat ion ), u nd ergo a pop to sis,and /or lo se the ir ab ili ty to ho me to the bo ne ma rrow mi-croe nvi ro nmen t.ME A SUR ING SO LU BLE PR OTEINNE T WOR KS IN H EM ATO PO IETICPR OG EN I TO R C UL TUR ESBio log i cal s yste ms are d yna mic, co nst ant ly re spon d-ing to mic roen viro nme ntal cue s in an at t empt to main -tai n an orga nism ’ s i nte rnal h ome osta sis. As re vie wedabo ve, the reg ul atio n of thi s co mple x en vi r o nm ent is d ue ,in l arge part, to t he e f fe cts o f prot ein mo du lato rs of ce llfun ctio n in tera ctin g wi th o ne a not her i n co mpl ex n et-work s (Fig. 1 ) t o prom ote vari ous c ell ul ar respo nses, in -clu din g pro life r a tio n, differe nti at ion , and su rviv al. B e-cau se c yto kin es are sec reted by sp ecifi c su bpo pu lat ion sof ce lls i n th e he tero gen eou s cel l p op ula tio ns ty pi cal ofhem ato po ies is, ba lan cin g th ese in t eract ion s requ ires di-rect or i nd irect fee db ack co nt rol mec han isms (13 0). Infact, us ing in v itro m od els of i n v i vo h e mato po iesis , ithas be en co nv inci ng ly sho wn t hat m atu re ce ll p opu la-tio ns in t he hem ato po iet ic co mpa rtmen t, o r th eir prod -uct s, di rectl y i mp act H SC resp on ses (99, 131 – 1 33). Al ongwith th e rec ent o bse rvat ion s tha t parti cul ar sub pop ul a-tio ns of cel ls p ot ent iate in v itro HS C e xp ansi on (1 34 ) an deng raftme n t (1 35 ), t hese fin din gs s ugg est t hat c urrentHSC c ult ure sy stem s ma y be in flu ence d by t he p rod uc-tio n o f m atu re cel ls cap ab le of s ecreti ng cyt ok in es, wh ich ,in tu rn, pro vid e reg ula tory si gna ls t o t he ce lls rem ain ingin c ult ure. T he p ub lish ed diffe r e nce s in t he se creti o n o fbo th p osi tiv e (e.g ., F lt-3) an d n eg ativ e (e.g ., TGF-b) reg -ula tors o f he mato po ie sis by subp op ula tio ns of p roge ni-HSC EXPANSION485FIG. 1 . Cyto k ine ne twork s r egu late b iolo gic al sy stem s. Th e ope n a rrow s ind icat e poss ible sou rces of cy tok ine s, wh ich ca nbe en do gen o us (p ara crin e o r au toc rine ) or e xo gen ous . So lid a r-rows sho w t ypi cal in tera cti ons tha t may occ ur b etwe en cy to-kine s p rese nt in a sys tem. The s e in tera ctio ns may b e res pon si-ble fo r reg u lati ng a var iety o f ce llul ar resp on ses in c lud ingpro lif era tion , dif fe ren tiat ion , an d ap opt osi s. tors an d m atu re c ells (9 8), as wel l as st romal c ells (13 6)furth er su ppo rts th e ex iste nce of t hese re gul ato ry mech -ani sms.On t his b asis, i t is c lear th at elu cid atin g t hese cyto ki nenet work s wo uld g reatl y ad d to t he u nd erstan di ng o f t hebio lo gi cal mech ani sms tha t re gul ate cel l r e spo nses invit ro—in format ion th a t cou ld t hen be u sed in d esig nin gHSC exp ansi o n c ult ures. Un fortu nat ely , u nti l recentl y , ithas b een ve ry d ifficu lt t o me asure t he p rote in sec retio npro files o f defi ned p opu lat ion s o f cell s i n th ese mix edcel l cu ltu re sy stem s. To t his e nd, o ur l ab ha s a dap ted ano vel qu ant itat i ve an aly tica l sy ste m (13 7,1 38 ) c apa ble o feffici ent ly an aly zin g cy tok in e se creti on i n t he co nt ext ofcel l-surfac e a nti gen ex press ion (phe not yp e) from hum ancord blo od prog eni tor ce lls usin g fl ow cyt ome try (Fi g. 2;Ma dlam b aya n et a l., in prep arati on ). In format ion b ein ggat here d usi ng t his a ssay i ncl ude s th e t yp es and q uan ti-tie s o f cyt ok ine s b ein g se crete d, a s well a s th e ph eno ty peand fun cti on (u sin g flo w c yto metri c so r t ing ) of c ells re-spo nsib l e fo r sec retin g t hese cyto ki nes. It is ou r g oal touse t his ass ay t o i d ent ify p hen ot ypi call y t ho se ce lls th atsecre te in hib ito ry cy to ki nes an d th us det ermin e th e ef-fects o f rem ovi ng th ese ce lls o n t he ex pa nsio n of t rans-pla nta b le HSC s. One l ine o f proo f t hat th is a ppro ach m aybe f e asib le co mes from stu die s i n wh ich bl ock in g an ti-bo die s o r ago ni sts spec ifi call y d irect ed ag ain st in hib ito rycyt ok in es h ave b een suc cessfu l in rev ersin g o r p reve nt-ing th e effect s of know n in hi bit ors su ch as T GF-b, MC P-1, an d MIP - 1a(116 ,11 7 ,13 9).BIOP R OC ESS D EV ELO P ME NTWe a re c urren tly in ves tig atin g th e “c ont roll ed” est ab-lish men t of cu ltu re co nd iti ons that fav or part icu lar invit ro hem ato poi eti c c ult ure e nd p oin ts by te stin g t he h y-po the sis t hat th e mani pu lati on o f in v itro p opu lat ion d y-nam ics, b y ch ang ing th e ty pes o f ce lls a nd cy to kin es p r e s-ent i n th ese c ult ures, will p rovi de a me ans o f e stab lish -ing cell ula r micro env i ronm ent s t hat fav or HSCself-ren ewal ov er d ifferen tia tio n. By c omb ini ng cy to ki nesup ple me nta tio n with p hen oty pic se lect ion a nd med iu mperfu sio n (which allo ws fo r th e di lut ion o f inh ibi tory au -toc rine facto rs), we a r e d evi sing a cu ltu re s yste m whe rewe ha ve con trol o f b oth exo gen ou s an d en do gen ous fa c-tors in h ema top oie tic p r o ge nit or c ell cu ltu res. In p reli m-ina ry st udi es, l ine age ne gat ive (Li n2) cell s were ob tai n edfrom UCB , c ult ured for 4 day s in me dia co nt aini ng TP O,SCF, a nd FL, resel ecte d to rem ove su bpo pu lati on s of m a-ture cel l t ype s, an d c ult ured fo r an a dd itio nal 4 d ays; th eyexh ib it ed a hi ghe r HSC co nte nt t han p arall el cult ures ofLin2cell s left in t he sa me cu ltu re con dit ion s fo r 8 day s(Mad lam b ayan , Rog ers, e t al. , un pub lis hed d ata).Fina lly , t he d eve lop ment o f bi op roces ses t o ex pan dHSCs to th erape uti call y releva nt q uan titi es req ui r e s th atcul ture s targ etin g th e g rowt h of H SCs b e adap ted t o clin -ica lly rele van t s cale s. St irred s usp ensi on c ult ure sy stems ,offeri ng th e ad va ntag es o f s cala bil ity a nd r e lat ive si m-pli cit y, a lon g wi th t he p ossi bil ity of in corp orati ng on lin edev ice s for med iu m e xch ang e a nd p erfusio n a nd c ell se-lec tio n (1 40 ) , are an at tract ive targe t t o ac hi eve thi s endgo al. Th e first us e of a stirred su spe nsio n cul ture sy ste mfor he mato po ieti c pro gen ito rs wa s d escrib ed by Sardo ni niand W u (14 1); h ere h uma n marro w cel ls we re ex pan ded10 - to 2 0-fol d o ver in pu t v a lue s whil e main ta ini ng pro-gen ito r cel l num bers. Sub sequ ent st ud ies u sing a si mil arsyst em sho wed th at by in crea sing i nn ocu lum d ensi ty andsup ple me nti ng t he med i a with a mo re ro b ust cy to ki necom bin at ion , C FC a nd L TC-IC n umb ers co ul d be ex -pan ded 66 - and 9-fo ld, resp ecti vel y (142 ). Sti rred su s-pen sio n cu ltu res ha ve a lso be en use d to am pli fy p rog en-ito r p opu lat ion s fro m CB and P B s ampl es, wh ere, fo rexa mpl e, 50 - to 10 0-fo ld ex pan sio ns of CFC an d 5- to15 -fold exp an sio ns of LTC -IC h ave b een repo rted in cu l-ture s ini tia ted w ith U CB C D341cell s (1 43). Sim ilarl y,Co llin s et al. (144 ) ha ve dem onst rated succ es sful ex pan -sio ns o f PB a nd UCB M NCs as well as PB CD3 41cell sin sti rred c ult ures usi ng b oth se rum-co nt aini ng an dserum -free m edi a.CO N CLU SION SSucc essful in v itro HSC ex p ans ion li kel y re qui res t h eabi lit y to co ntro l d yn amic al ly b ot h po siti ve a nd n ega tiv efacto rs prod uce d d urin g i n v itro he mato po ieti c ce ll cu l-ture . For th is in format ion to b e use ful, a co nn ecti on mu stbe mad e b etwe en cell id ent ity (usi ng c ell -surface mark -ers) an d t hos e secret ed pro du cts t hat are c apab le o f in -terac tin g wi th HSC s. Ult imat ely , the d eve lop men t of ef-fici ent HSC c ult ure stra tegi es will requi re co mbi nin gMADL AMB AYAN ET AL .486FIG. 2 . Th e g el mi cr odr op ( GMD) as say (13 7,1 38) . Th is as- sa y is ca pab le of e ffic ien tly ana lyzi ng cyt oki ne se cre tion f romind i vid ual cel ls i n the con tex t of c ell -s urfa ce ant ige n e xpr es-sio n . We a re us ing t his a ssay to ch ar act eriz e d yna mic ch ang e sin th e cyt okin e sec ret ion pro file s o f spe cifi c sub pop ula tion s o fhe ma topo iet ic pr oge nit ors ( bas ed o n th e ir c ell -sur fac e ph eno -typ e ) du ring in vitr o c ultu re. cyt ok in e sup ple men tati on re gime n s with th e rem ov al o fspe cific cell types as th ey e merge . Thi s may add iti on allyall ow fo r th e ge nera tio n o f c ult ures ca pab le o f s peci ficfun ctio n al ou tpu ts (i .e., co nta ini ng ce lls re qui red for t heimme dia te su pp ort o f the p ati ent s wit h co mpro mise d i m-mun e syste ms, or cell s t hat fa cili tat e HSC ho min g). Gen-erati ng , s uch “de sign er tran spla nts” will requ ire no t o nlythe iden tifi cati on o f con dit ion s ca pab le of amp lify ingHSC nu mbers, b ut al so no vel a pp r o ach es for con tro llin gcel l po pul ati on flu xes in the se mix ed cel l cu ltu res.AC KNO WLE D GM EN TSThi s wo rk w as sup po rted b y a Na tural Scie nce s andEng in ee r in g R esearc h C ou nci l (NSER C ) of C ana da g rantto P.W.Z . G.J.M . h ol ds a NSERC po stg radu ate fel low-shi p.RE F ERE NC ES1. Ca iro MS a nd JE W agn er. (1 997 ). Plac enta l an d/ o r um -bi lic al cor d blo od: a n a lte rnat iv e so urce of hema top oie ticste m cel ls fo r tr ans plan tat io n. Bloo d 90 :46 65– 4 678 .2. W agn e r JE, NA Kern an, M Ste inb uch , HE Brox meye r a ndE Glu ck man. ( 199 5). All oge nei c s ibli ng umbi lica l-c ord -bl oo d t rans pla nta ti on in c hild ren with m alig nan t an d n on -mal ig nan t disea s e. La nce t 3 46: 214 – 2 19.3. W agn e r J E , J Ro sen tha l, R Swee tman , XO Shu , SMDav ie s, NK Ra msay , PB Mc Glav e, L Se nde r an d MSCair o . (19 96) . Succ ess ful tra nsp lan tati o n of H L A -mat ch ed an d HLA- misma tch ed u mbili cal c ord b lo od fro mun re late d d o nors : an aly sis of e ngr aftme nt a nd a cu te gr aft -ve rs us- hos t d isea se. Bl ood 88 :79 5– 80 2.4. Dr yga ls ki A, G Xu , D Co nsta nti nes cu, I Kas hiwa kur a, TFarl ey , L Do bri la, P Rub inst ein an d JW Adams on. (2 000 ).The fr equ enc y a nd pr olif erat ive pote nti al of mega kar y-oc y tic co lon y- fo rmin g cel ls (Me g-CFC) i n cor d b loo d ,cy to kin e-mo bili zed pe rip he ral b loo d and b on e ma rro w,an d t hei r c orr elat ion with t otal CFC nu mbe rs: im plic a-tio n s fo r the qu ant itati on o f Me g-CFC to pr edic t pl ate leten g raft ment foll owin g c ord b loo d tran spl anta tio n. Bo neMar ro w Tra nsp lan t 25: 102 9– 10 34 .5. Gl uck ma n E, V Roc ha , A Bo yer -Cha mma rd, F Loc atel li,W A rces e, R Pasq uin i, J Ort ega , G S o uil let, E Ferre ira ,JP La por te, M Fern and ez an d C Chas tan g. (1 997 ). Out -co me o f co rd- blo od tra nsp lan tati on fr om r elat ed a nd un -re la ted don ors . Eu roc ord Tra nsp lan t Gr oup and the Eur o-pe an Bloo d an d Mar ro w Tran spl anta t ion Gr oup . N En glJ Me d 33 7:3 73– 3 81.6. W eke rl e T an d M S y ke s. ( 200 1). M ixed c hi meris m andtra n spla nta tion to ler an ce. A nnu Rev Me d 52 :353 – 3 70.7. Br aze lt on TR, FM Ro ssi , GI Kes het a nd HM Blau . (2 000 ).From ma rrow to b rain : exp res sion o f n euro na l p hen oty pesin a d ult mi ce. Sci enc e 2 90 :177 5– 17 79 .8. Gu sso n i E, Y So neo ka, CD Stri ckl and , EA Buzn ey, MKKha n , AF Fli nt, LM Ku n kel and RC Mu llig an. (1 999 ).Dys tr oph in exp ress ion i n t he m dx mous e res tore d b y s temce ll tra nsp lan tati on. Na tur e 4 01: 390 – 34 0.9. La gas se E, H Co nno rs, M Al -Dha limy, M Rei tsma , MDoh s e, L Osb orn e, X Wa ng, M Fin ego ld, IL Weis smanan d M Gr ompe . ( 2 000 ). Pur ifie d h ema top oie tic s tem ce llsca n d iff eren tia te in to h epa toc ytes in v ivo . Natu re M ed6: 12 29 – 1 23 4.10. An de rson DJ, FH Gage an d I L We is sman . (2 001 ). Canste m ce lls c ross lin eag e b oun da rie s? Nat ure Med7: 39 3– 39 5.11. Ko pe n G C, DJ Prock op an d DG Phin ney . (19 99) . Mar -ro w str omal cel ls m igra te t hro ugh ou t fo reb ra in a nd cer e-be ll um, a nd the y dif fer enti ate int o a stro cyt es af ter in jec -tio n i nto ne ona tal mo u se bra ins . Pro c Natl A cad Sc i USA96 :1 07 11– 1 071 6.12. Kr aus e DS, N D Th eise , MI Col lec tor, O He neg ari u, SHwan g , R Ga rdn er, S Ne utz el an d SJ Sh ar kis . (2 001 ).Mul ti -org an , multi- lin eag e eng raft ment by a sing l e b onemar ro w-de riv ed st em cel l. Ce ll 10 5:3 6 9– 37 7.13. Ga bu tti V, F Timeu s, U Ra meng hi, P Sara cc o, D Fas sio ,MC Giu bel lino , R M inie ro a nd E Mad on. (1 993 ). Humanco rd bl ood pro gen itor s: ki net ics, r egu lati on and th eir usefo r he mopo iet ic rec ons titu tion . Bo ne Marr ow Trans pla nt12 (Supp l 1) :84– 8 6.14. Gl uck man E, HA Br oxme yer , AD Au erb ach , HS Frie d-man , GW Do ug las , A De ver gie , H Es per ou, D Thi erry , GSoc ie , P Le hn et al. (19 89 ). He mato poi etic re con sti tuti onin a pat ient with Fa nco ni’ s an emia by me ans o f um bili -ca l- cord bl ood fro m a n HLA- iden tic al sib ling . N En gl JMed 3 21:1 17 4– 1 180 .15. Br oxm eye r HE, GW Dou gla s, G Han go c, S Co ope r, JBar d, D Eng lish , M Arn y, L Th omas an d EA Boys e.(1 98 9). Hu man u mbili cal cor d b loo d as a po te nti al sou rceof tr ans plan tab le h emat opo ieti c s te m/ pr oge nit or c ells .Proc Na tl Acad Sci USA 8 6:3 828

Baby Love Birth Center

Welcoming all new babies born at the Baby Love Birth Center!

Amelia Paige

Posted by Baby Love Birth Center 1319 Baby Love Birth Center Baby Love Birth Center has not set their biography yet User is currently offline on Thursday, 30 August 2012 in Birth Stories Tweet

Copied from The Duffy Family Blog


On Wednesday, August 22, 2012 I woke up around 10am. The last few nights I had a hard time sleeping due to my hips being sore and waking up to go to the bathroom so often. This night I finally got some great rest and I am so thankful for it.

While laying in bed that morning I started to feel some cramps and what I thought were braxton hicks. Nothing to be concerned about since I knew it was just one of the things that prepares my body for the big event. My mom invited me to lunch and I told her at noon that I started timing my braxton hicks since they were coming so often. They were 2-3 minutes apart lasting for about a minute. I thought this just had to be braxtons since nobody starts labor at this rate. My mom even said it probably was pre-labor or false labor since they didn't start at the typical 10-15 minutes apart. Plus they really were not painful. So I just continued to watch TV. My mom arrived at my house to pick me up for lunch at about 12:30pm and I told her they were getting stronger. They still just felt like period cramps with my stomach tightening like a braxton hicks, but not painful enough to convince me that I was in labor. Since I wasn't feeling up to going e5c out, my mom and I just went through the drive thru at McDonald's so I could get a smoothie and something to eat. When we got back to my house she convinced me to text Bryan to have him come home and skip the meeting he had after school. He had guessed she would come the following day. So we kept joking that maybe this meant he would be right. My mom then left around 1:30pm and Bryan got home about 2:30pm. When he got home I was bouncing on my yoga ball listening to the playlist of music I made for myself during labor. At this point I had to stop during a contraction and breath. I could still talk through them, but I had to focus a little more. We were not too concerned so I kept bouncing while Bryan made something to eat.

At about 4:00 Bryan called a client of mine to cancel my consultation I had scheduled that night. I thought about calling them myself, but since my contractions were so close together I would probably not sound the way I should when talking to a client. My mom came to pick up the dog so he didn't annoy me and she called the birth center to give them a heads up that I may be coming in sometime in the future. They didn't seem too concerned since I was still barely convinced myself that this was the real thing. I could still talk through the contractions. I just closed my eyes and bounced on my ball.

I texted my doula Crystal and we decided it would be nice for her to come over even if this wasn't real labor. She arrived at my house about 5:30pm. She came and sat on the edge of my bed while I was still on my yoga ball. She watched me go through some contractions and said to me that this may be false labor, but that we should do some things to see if we could get things moving. So the three of us headed outside to do some walking. We walked two blocks in my neighborhood. During a contraction I would hold on to Bryan and sway my hips. A few times Crystal had me try walking through a contraction. Some were not as intense as others so I could do it. At this point I started to feel more convinced that this was the real deal. Since the contractions were not intense for that long (maybe 30 min at this point) I thought I had a long night ahead of me. At about 6:15pm Crystal called the birth center when we got back home. She told them my contractions were about 2-4 minutes apart lasting for only about 30 seconds. The midwife said she would like to see me have contractions that lasted for 2 minutes long. So she suggested I take a bath and try to relax some more. Bryan started the tub and we all laughed at our funny tub that is oddly sunk into the ground at our house. I got in the tub and could not get comfortable. I felt my belly was just too big to fit into the water like I wanted. A few minutes later I had a contraction that gave me the urge to push. It wasn't an intense feeling, so I told Bryan and Crystal I had the urge but didn't know if it was real. Crystal looked at Bryan in a panic and told him to immediately call the birth center and tell them we were coming. She helped me out of the tub and I dried off real quick so that I could make it back to my yoga ball before the next contraction hit. I kept telling them I really hope Samantha (my midwife) doesn't send me home because I am only like 3 cm dilated or something similar. I was so worried I would drive all the way there and be too early.

Bryan helped me in the car and we started on our way. I had a 815 bout 4-5 of the most intense contractions yet in the car. Since I had a slight urge to push I had sooo much pressure down below. It hurt to sit so I kept raising myself off the seat with the handle above the door. At this point I couldn't calm myself and I was pretty much screaming. Bryan kept calm and drove as smoothly as possible even though I got mad at him for every little bump in the road. I look back now and feel bad and can't believe how he managed next to me.

We arrived at the birth center at about 7:00pm and we went straight back to the Angel Room suite that we chose to deliver in. Samantha had already started the tub for me. She asked me if I wanted to be checked and I said yes. As she was checking me I was just waiting for the bad news. She looked up at me and smiled saying you're 9 cm dilated! I was in shock! I looked at Bryan and was so excited to know that I was already almost done. The tub wasn't full yet but I asked to get in right away. I believe I had one more contraction in the tub and then started feeling a extreme urge to push.

The contractions were very different now. Not necessarily painful. They now took over my body making me push so hard that I couldn't fight back. Bryan put his bathing suit on and hoped in the tub to support me. I grabbed onto the handle above my head and started to groan with each push that took over me.

I laugh now because I remember hearing Bryan push right along with me. It encouraged me to know he was doing everything he could to support me. My doula stayed with me the entire time lea 1f5e ning over the tub massaging my legs. Samantha would come in every once and a while to check on me and listen to the baby's heartbeat. No one ever told me what to do, how to push or when to push. They just let my body do what it was made to do. This is one of the many reasons I chose to birth at Baby Love Birth Center, rather than a hospital. My water even broke on its own about a half hour into pushing and it looked like a balloon burst in the water.

I pushed for about an hour and a half until Amelia was born at 8:35pm. Once the cord stopped pulsating, Bryan cut it. The placenta came about 15 minutes later and we thought it was pretty cool to see in person. I was in complete shock that I had just had a baby!!!! I had finally convinced myself I was in labor only about 3-4 hours ago and she was already in my arms! Since she was born in such a calm atmosphere and in the warm water which was familiar to her she was very calm. She only cried a little bit to get the stuff out of her mouth, but since there were no drugs of any kind in our systems she was wide awake and looking around. We couldn't get over her chunky cheeks, I was so in love.

My parents arrived and came to see Amelia shortly after she was born. My mom got emotional which then made me even more emotional. Samantha waited until we were ready for her to take Amelia to be dried and weighed. I got out of the tub and into bed. Amelia was then shown off to the rest of the family in the waiting room outside my door. Bryan brought her back to me and my mom told me that I just had a baby that was 8lbs 14oz! I was in shock and couldn't believe I had such a big baby. Later the family came into my room with cake to sing happy birthday to Amelia.

A BIG thank you to...

Stacy from Set Free Photography who took all of my birth photos above!

Baby Love Birth Center and it's staff Samantha McCormick and Alexis

Crystal Alejo who was my doula

Jaci Sieben who taught the HypnoBirthing class I took through Bonita Birthing Solutions (offered at Baby Love)

and Mama's Chiropractics for helping my body be prepared.

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