Pluripotent Hem opoietic Stem Cells in Murine Postmortem Bone Marrow

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1 ANNALS OF CLINICAL A N D LABORATORY SC IENCE, Vol. 10, No. 1 Copyright 1980, Institute for Clinical Science, Inc. Pluripotent Hem opoietic Stem Cells in Murine Postmortem Bone Marrow PAUL I. LIU, M.D., P h.d.,* KAM C. PO ON, P h.d.,* LYNN CROOK, M.D., P h.d.,* C H O U C. H O N G, DVM, P h.d.,* and M ITSUOKI E G U C H I, M.D., PH.D.f Departments o f Laboratory Medicine* and Pathology, f Medical University o f South Carolina, Charleston, SC ABSTRACT A lthough 12-hours postm ortem m urine bone m arrow cells exhibit extensive degeneration, th ese cells w hen infused into irradiated m ice produce erythrocytic, granulocytic, m egakarocytic an d m ixed colonies in th e ir spleen. T hese observations clearly dem onstrate the p resence o f p luripotent hem o p o ietic stem cells an d th eir p roliferative capability in 12-hours postm ortem, m urine bone m arrow. T hese observations suggest th at cadaveric bone m arrow transplantation m ay be possible in patients w ith hem atologic disorders. Introduction Simonsen s Laboratory, Gilroy, CA. In our previous report,2 evidence was p resented that proliferative function persists in hum an and m urine cadaveric bone marrow. U tilizing m ethylcellulose clonal cell culture assays, erythropoietic burst and granulopoietic colony form ation from bone m arrow cells, collected as late as 19-h o u rs p o stm o rte m w as o b se rv e d. Postm ortem persistence of hem opoietic stem cells in m ice as assayed by spleen colony tech n iq u e p aralleled that of the com m itted precursors assayed in clonal cultures. In this com m unication, ultrastru ctu ral, histological an d cytological ev id en ce are p re se n te d for th e plurip o ten t n atu re o f th e m urine cadaveric spleen colony form ing cells. M aterials and M ethods P r e p a r a t i o n o f D o n o r B o n e M a r r o w C e l l s T he anim als u sed w ere m ale B D F m ice w eighing 25 to 30 g.* M ice w ere killed by cerv ical d islo catio n an d k e p t at room tem perature. Sam ples of fresh and 12- h our postm ortem bo ne m arrow cells suspensions w ere collected from nine m ice by flu s h in g th e fe m u r w ith 1 m l o f a-m edium by m eans of a # 2 3 gauge n e e dle attached to a syringe. Single cell suspen sio n s w ere sep arated into sam ples c o n ta in in g 2 x c e lls p e r m l o f / $00.90 Institute for Clinical Science, Inc.

2 S T E M C E L L S IN P O S T M O R T E M BON E MARROW a -m e d iu m for O-hr p o stm ortem group and 8 X cells p e r ml o f a -m e d iu m for 12h o u rs p o stm ortem group, resp ectiv ely. I n V iv o A s s a y f o r M u r i n e H e m o p o ie tic P r e c u r s o r s M u rin e h e m o p o ie tic stem cells w ere assayed usin g th e in vivo sp le e n colony te c h n iq u e d e v e lo p e d b y T ill a n d M cc u llo c h.4 O n day 1, 40 m ice w e re irra d ia te d w ith 900 rads (250 KVP G E maxitron units at 30 ma a n d a q u arte r-c o p p er filtratio n ).! O n day 2, the irra d ia te d m ice w e re in fu s e d via th e ir tail v e in s w ith e ith e r 12-hours p o stm o rtem m u rin e m ar row cells (24 m ice) or fresh m u rin e m ar row cells (11 m ice). T h e re m a in in g five i r r a d ia t e d m ic e w e r e i n j e c t e d w ith a -m e d iu m only for th e control purpose. On day 10, th e m ice w e re sacrificed. T h e ir s p le e n s w e re re m o v e d a n d d is s e c te d. N u m ero u s im p rin ts o f th e sp leen s w ere stain ed w ith W rig ht s stain. S p leen s w ere th e n fixed in B o u in s so lu tio n an d serial sectio n s o f th e e n tire sp le e n w e re sta in e d w ith hem atoxylin a n d eosin. U sing the im prints an d H & E sta in e d serial h isto logic sections o f sp leen s, th e n u m b e r of sp le e n colonies w ere co u n te d an d clas s if ie d as e r y th r o c y tic, g r a n u lo c y tic, m egakaryocytic an d m ix ed according to th e ir p rin cip al h e m o p o ie tic cell lines. For ultrastru ctu ral study, in d iv id u a l colonies w ere rem oved from th e sp leen s u n d e r a d is s e c tin g m ic ro s c o p e. T h e s e tis s u e s w ere p re p a re d for tran sm issio n electro n m icroscopy by fixation in 3 p e rc e n t glutara ld eh y d e a n d p ro c essed ro u tin ely. 41 m ortem b o n e m arrow cells. T h e sp le e n of th e n o n -irrad iated m ice, w h ich w e re in fu se d w ith a -m e d iu m alone, show s no sp le e n colonies (figure 2). Irra d ia te d m ice w ho do n o t receiv e m arrow tran sfu sio n s d ie d w ith in five days to a m onth, an d a u topsy o f th e se m ice rev ealed no id e n ti fia b le h e m o p o ie tic c o lo n ie s in t h e i r s p le e n s. C o n v e rse ly, m ost o f th e irra d ia te d m ice w ho re ceiv ed e ith e r fresh or 12-hours p o stm ortem m arrow transfusion h a v e s u rv iv e d m o re th a n six m o n th s. C o m p ared to fresh m arrow cells, th e 12hours postm ortem m arrow cells w ere a p proxim ately 25 p e rc e n t as e ffic ie n t in pro- *4 A ft'm '. 4 R esults In figure 1 is illu stra te d th e low m ag n ific a tio n lig h t m ic ro sc o p ic p ic tu re o f h em o p o ietic colo n ies in a m ouse sp leen w ho re ceiv ed w h o le body irrad iatio n fol lo w ed by an in fu sion o f 12-hours postt Courtesy of the D ivision o f Radiotherapy, M edi cal U niversity of South Carolina. 1 2 F i g u r e 1. A s e c tio n o f s p le e n fro m a n ir r a d ia te d m o u s e in f u s e d w i th 1 2 -h o u rs p o s tm o r te m b o n e m a r ro w c e lls s h o w in g m ic r o s c o p ic h e m o p o ie tic c o lo n ie s (H&E sta in X 9). FIG U R E 2. A s e c t i o n o f s p l e e n f ro m a n o n i r r a d i a t e d m o u s e i n j e c t e d w ith a - m e d i u m o n ly s h o w in g a b s e n c e o f c o lo n ie s ( H&E s ta in x 9).

3 42 L IU, P O O N, C R O O K, H O N G A ND E G U C H I F i g u r e 3. An im print of the m ixed colony show ing a megakaryocyte (m) and num erous granulocytic cells (Wright s stain x 700). F i g u r e 4. An im print o f th e granulocytic colony show ing num erous im m ature granulocytic cells (W right s stain x 700). L L ft# # M 9» * ^ m -./ ' ' V $ f i! d u c in g s p le e n c o lo n ie s.2 T h e se s p le e n co lo n ies co n sist o f d iffe ren tiated h e m o p o ie tic c e ll lin e s su ch as ery th ro cy tic, g r a n u lo c y tic a n d m e g a k a ry o c y tic o r m ixed cell lines. Im p rin ts of the sp le e n co lo n ies d em o n strate m ixed (granulocytic a n d e r y th r o c y tic o r g ra n u lo c y tic a n d m e g a k a ry o c y tic ), g r a n u lo c y tic a n d ery th ro cy tic colonies as show n in figures 3, 4 an d 5. T h e erythrocytic cells in th e ery th ro cy tic co lo n ies m ay b e m is in te r p r e te d as ly m p h o c y te s in r o u tin e hem atoxylin an d eosin sections. m TABLE I Cytohistological Classifications of Spleen Colonies Derived from Cadaveric and Fresh Murine Bone Marrow Cells 5 > F i g u r e 5. An im print of th e erythrocytic col onies showing nucleated erythrocytic cells (W right s stain x 700). Colonies (Percent) Fresh Marrow Cells 12-Hour Postmortem Ma r r o w Cells Erythrocytic Granulocytic Mixed Megakaryocytic

4 ST E M C E L L S IN P O S T M O R T E M B O N E M A RROW 43 FIGURE 6. A n e r y th r o c y tic c o lo n y o f s p le e n d e r i v e d from in f u s io n o f 1 2 -h o u r p o s tm o r te m m u r in e b o n e m a rro w c e lls s h o w in g e r y th ro c y tic c e lls w ith c h a r a c te r is tic c h r o m a tin p a t te r n (H & E s ta in X 1,200). F i g u r e 7. E le c tr o n m ic r o g ra p h o f e a r ly a n d la te e r y th ro b la s ts in a s p le e n c o lo n y ( x 3,0 0 0 ).

5 44 LIU, POON, CROOK, HONG AND EGU CHI How ever, ultrastructural exam ination o f erythrocytic colonies and hig h er m agnification of W right s stain on im print of erythrocytic colonies dem onstrated the p resen ce o f ch aracteristic ery th ro cy tic cells in various stages of developm ent. T hese cells are dem onstrated in figures 6 and 7. T he percentage of various types of colonies in the postm ortem and fresh m arrow cells are p resented in table I. In both sam ples, about 33 p ercen t o f th e colonies could be classified as m ixed colonies. T hese data agree w ith the previous work reported by Curry and T re n tin 1 and indicate that postm ortem bone m arrow cells p o ssess p lu rip o te n t h e m o p o ietic stem cells. D iscussion Recently, allogenic bone m arrow transp la n ta tio n h as e m e rg e d as a v ia b le therapeutic approach in various hem atologic disorders.4 O ne major lim itation is that the successful transplantation is lim ited m ostly to transplantation b etw een h isto c o m p a tib le sib lin g s. In o rd e r to exam ine the possibility th at cadaveric h u m an b o n e m arro w c e lls c o u ld b e utilized for bone m arrow transplantation b etw een u n related individuals, the proliferative function of hum an and m urine c a d a v e ric b o n e m arro w c e lls w as exam ined by us. Significant postm ortem survival of hem opoietic functions w ere o b se rv e d.2 H o w ev er, w h ile th e p o s t m ortem survival of m arrow function has b een dem onstrated by us, w e have not p ro v en th e p lu rip o te n t n a tu re o f the cadaveric sp leen colony form ing cells. U ltrastructural and cytological analysis of th e sp leen co lo n ies d e sc rib e d in this p ap er confirm s th e postm ortem persisten ce of p lu rip o ten t h em opoietic stem cells. E s ta b lis h m e n t o f b o n e m arrow banks to store cadaveric hum an bone marrow cells may broaden the indications for b one m arrow transplantation and provide m eth o d s to p ro cure larg er am ounts of histocom patible bone m arrow cells in the future. Acknowledgm ent T his in v estig atio n w as su p p o rte d by South Carolina State Biochemical Research Grant. Assistance was provided by Joyce Christopher, Nancy Trent, Jim Burkette and Janie R. Dingle. R eferences 1. C u r r y, J. L. and T r e n t i n, J. J.: Hemopoietic spleen colony studies. I. Growth and differentiation. Develop. Biol. i5: , L iu, P. I., O g a w a, M., C r o o k, L., O c h ia, R., and U p s h u r, J. K.: Proliferative function of cadaveric bone marrow cells. Amer. J. Hemat. 5: , T h o m a s, E. D., S t o r b, R., C l e f t, R. a., F e f e r, A., J o h n s o n, F. L., N e im a n, P. E., L e r n e r, K. G., G l u c h s b u r g, H. a n d B u c k m a n, C. D.: B o n e m a rro w tra n sp la n ta tio n. N e w E n g. J. M e d. 292: a n d , T i l l, J. E. and M c C u l l o c h, E. A.: A direct m easurem ent of the radiation sensitivity of normal mouse bone marrow cells. Rad. Res. 14: , 1961.