Protection and Repair of the Nigrostriatal Pathway with Stem-Cell-Derived Carotid Body Glomus Cell Transplants in Chronic MPTP Parkinsonian Model

Ci a ion: Villadiego, J.; Muñoz-Manchado, A.B.; Sob ino, V.; Bonilla-Henao, V.; Suá ez-Luna, N.; O ega-Sáenz, P.; Pa dal, R.; López-Ba neo, J.; Toledo-A al, J.J. P o ec ion and Repai o he Nig os ia al Pa hway wi h S em-Cell-De i ed Ca o id Body Glomus Cell T ansplan s in Ch onic MPTP Pa kinsonian Model. In . J. Mol. Sci. 2023,24, 5575. h ps:// doi.o g/10.3390/ijms24065575 Academic Edi o : Massimo Conese Recei ed: 15 Feb ua y 2023 Re ised: 10 Ma ch 2023 Accep ed: 11 Ma ch 2023 Published: 14 Ma ch 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). In e na ional Jou nal o Molecula Sciences Communica ion P o ec ion and Repai o he Nig os ia al Pa hway wi h S em-Cell-De i ed Ca o id Body Glomus Cell T ansplan s in Ch onic MPTP Pa kinsonian Model Ja ie Villadiego 1,2,3,*,† , Ana B. Muñoz-Manchado 4,†, Ve ónica Sob ino 5, Vic o ia Bonilla-Henao 1,2,3 , Nela Suá ez-Luna 1,2,3, Pa icia O ega-Sáenz 1,2,3, Rica do Pa dal 1,2,3 , JoséLópez-Ba neo 1,2,3 and Juan J. Toledo-A al 1,2,3,* 1Ins i u o de Biomedicina de Se illa (IBiS), Hospi al Uni e si a io Vi gen del Rocío/CSIC/Uni e sidad de Se illa, 41013 Se illa, Spain 2Depa amen o de Fisiología Médica y Bio ísica, Facul ad de Medicina, Uni e sidad de Se illa, 41009 Se illa, Spain 3Cen o de In es igación Biomédica en Red sob e En e medades Neu odegene a i as (CIBERNED), 28029 Mad id, Spain 4 Unidad de Biología Celula , Depa amen o de Ana omía Pa ológica, Biología Celula , His ología, His o ia de la Ciencia, Medicina Legal y Fo ense y Toxicología, INiBICA (Ins i u o de In es igación e Inno ación Biomédica de Cádiz), Uni e sidad de Cádiz, 11003 Cádiz, Spain 5 Ins i u o Maimónides de In es igación Biomédica de Có doba, Depa amen o de Biología Celula , Fisiología e Inmunología, Uni e sidad de Có doba, 14004 Có doba, Spain *Co espondence: [email p o ec ed] (J.V.); [email p o ec ed] (J.J.T.-A.); Tel.: +34-955-923-034 (J.V.); +34-955-923-035 (J.J.T.-A.) † These au ho s con ibu ed equally o his wo k. Abs ac : An ipa kinsonian ca o id body (CB) cell he apy has been p o en o be e ec i e in oden and nonhuman p ima e models o Pa kinson’s disease (PD), exe ing ophic p o ec ion and es o a- ion o he dopamine gic nig os ia al pa hway. These neu o ophic ac ions a e media ed h ough he elease o high le els o glial-cell-line-de i ed neu o ophic ac o (GDNF) by he CB ansplan . Pilo clinical ials ha e also shown ha CB au o ansplan a ion can imp o e mo o symp oms in PD pa ien s, al hough i s e ec i eness is a ec ed by he sca ci y o he g a ed issue. He e, we ana- lyzed he an ipa kinsonian e icacy o in i o -expanded CB dopamine gic glomus cells. In as ia al xenog a s o a CB neu osphe es we e shown o p o ec nig al neu ons om degene a ion in a ch onic MPTP mouse PD model. In addi ion, g a s pe o med a he end o he neu o oxic ea men esul ed in he epai o s ia al dopamine gic e minals h ough axonal sp ou ing. In e es ingly, bo h neu op o ec i e and epa a i e e ec s induced by in i o -expanded CB cells we e simila o hose p e iously epo ed by he use o CB ansplan s. This ac ion could be explained because s em-cell-de i ed CB neu osphe es p oduce simila amoun s o GDNF compa ed o na i e CB issue. This s udy p o ides he i s e idence ha in i o -expanded CB cells could be a clinical op ion o cell he apy in PD. Keywo ds: Pa kinson’s disease; cell he apy; ca o id body s em cells; neu op o ec ion; neu o es o a ion 1. In oduc ion Pa kinson’s disease (PD) is cha ac e ized by he p og essi e dea h o dopamine gic subs an ia nig a pa s compac a (SNpc) neu ons p ojec ing o he s ia um. The degene a ion o nig al neu ons esul s in he deple ion o dopamine (DA) in he s ia um, con ibu ing o he ypical mo o symp oms o he disease. Cu en PD pha macological he apies aim a inc easing he s ia al DA inpu h ough he adminis a ion o le odopa (a DA p ecu so ), DA ecep o agonis s o inhibi o s o DA deg ada ion [ 1 , 2 ]. Fu he mo e, he ansplan a ion o DA-sec e ing cells o inc ease he s ia al DA con en in PD pa ien s has In . J. Mol. Sci. 2023,24, 5575. h ps://doi.o g/10.3390/ijms24065575 h ps://www.mdpi.com/jou nal/ijms In . J. Mol. Sci. 2023,24, 5575 2 o 12 been in ensi ely in es iga ed. Among he di e en dopamine gic issues es ed, he bes clinical bene i was ob ained wi h allog a o human e al en al mesencephalic issue (h VM) in open-label ials [ 3 – 5 ]. Howe e , wo double-blind con olled ials ques ioned he clinical e icacy o his p ocedu e, and showed he appea ance o dyskinesias in some g a ed pa ien s [ 6 , 7 ]. The lack o a clinical imp o emen and he side e ec s obse ed in he double-blind con olled ials we e a ibu ed o me hodological issues, such as pa ien selec ion, h VM issue p epa a ion and implan a ion and he immunosupp essi e egime applied pos - ansplan a ion [ 8 , 9 ]. Recen ly, a new open-label s udy, whe e hese me hodological issues ha e been s ic ly s anda dized, was designed o e-e alua e he clinical e icacy o h VM g a s [ 10 , 11 ]. In addi ion, o he an ipa kinsonian cell-based he apies using midb ain dopamine gic p ogeni o s gene a ed om emb yonic s em cells o induced plu ipo en s em cells (iPSC) a e cu en ly being es ed in clinical ials [12,13]. The ca o id body (CB) is an al e na i e dopamine gic issue used in an ipa kinsonian cell- he apy [ 14 ]. The CB is a bila e al o gan, loca ed in he ca o id bi u ca ion, ha con ains neu al c es -de i ed highly dopamine gic glomus cells, which ac as oxygen senso s ha elease dopamine in esponse o hypoxemia [ 15 ]. The in as ia al ansplan a ion o dopamine gic CB glomus cells was shown o p omo e signi ican eco e y in oden and nonhuman p ima e PD models [ 16 – 20 ]. Despi e he high dopamine con en o CB glomus cells, eco e y induced h ough CB g a s is belie ed o be mainly media ed by he elease o he glial-cell-line-de i ed neu o ophic ac o (GDNF), which exe s neu o ophic p o ec ion and es o a i e e ec s on he nig os ia al pa hway [ 18 , 19 , 21 ]. Due o unila e al su gical CB esec ion ha ing no signi ican side e ec s [ 22 ], wo pilo clinical ials e alua ed he sa e y and clinical e icacy o CB au o ansplan a ion, showing ha i can induce a clinical imp o emen in PD pa ien s [ 23 , 24 ]. Howe e , he small size o he CB and he po en ial damage o his o gan in PD pa ien s appea o be impo an limi a ions o he clinical ou come o CB au o ansplan a ion [ 24 – 26 ]. Du ing ecen yea s, ou labo a o y has desc ibed and cha ac e ized a popula ion o neu al c es -de i ed s em cells in he adul CB. These neu al s em cells a e he sus en acula o ype II cells in he o gan, which, upon exposu e o hypoxia, a e con e ed in o nes in + -p oli e a ing p ogeni o s and di e en ia e in o new dopamine gic glomus cells. Fu he mo e, an in i o cul u e o neu al CB s em cells gene a es clonal cell colonies (named neu osphe es) composed o a co e o p oli e a ing p ogeni o s su ounded by blebs o di e en ia ed dopamine gic CB glomus cells [27–30]. In his wo k, as p oo o concep , we es ed whe he s em-cell-de i ed dopamine - gic CB glomus cells exe neu o ophic p o ec ion o he damaged nig os ia al pa hway and, hus, amelio a e expe imen al pa kinsonism. We showed ha in i o cul u ed CB neu osphe es p oduced simila amoun s o he dopamino ophic ac o GDNF han na i e CB issue, and ha GDNF exp ession was main ained a e a s ia al ansplan a ion. Fu - he mo e, in as ia al g a s o CB neu osphe es induced neu op o ec i e and es o a i e ac ions in he nig os ia al pa hway o ch onic MPTP pa kinsonian mice. Rema kably, he neu o ophic ac ions exe ed by in i o -expanded dopamine gic CB glomus cells we e simila o hose p e iously epo ed o CB issue [ 19 , 31 – 34 ]. This s udy sugges s ha s em-cell-de i ed CB glomus cells could be a po en ial clinical op ion o cell he apy in PD. 2. Resul s 2.1. His ological Analysis and GDNF Exp ession o CB S em-Cell-De i ed Neu osphe es Adul CBs con ain quiescen mul ipo en neu al s em cells ha a e ac i a ed unde sus ained hypoxemia, inducing o gan g ow h. These neu al s em cells ha e been iden i ied as sus en acula ype II CB cells, which, unde hypoxia, change hei quiescen GFAP + s a e o a p oli e a i e nes in + pheno ype and, he ea e , di e en ia e in o ma u e dopamine gic CB glomus cells [ 27 ]. Mo eo e , CB neu al s em cells o m clonal colonies in i o ha esemble CB glome uli, wi h a co e o nes in + p ogeni o s su ounded by blebs o ma u e TH + glomus cells [ 27 , 28 , 35 ]. He e, we assessed he in i o cul u e o bo h mouse and a CB neu osphe es. As p e iously epo ed [ 36 ], he s uc u e o CB-de i ed neu osphe es is quali a i ely simila be ween bo h oden species, p esen ing p oli e a i e nes in + p o- In . J. Mol. Sci. 2023,24, 5575 3 o 12 geni o s and di e en ia ed TH + glomus cells (Figu e 1a,b). Howe e , a CB neu osphe es a e la ge , and showed a highe numbe o di e en ia ed TH + glomus cells in he blebs han mouse CB neu osphe es (Figu e 1b). Because he eco e y induced h ough CB cell he apy in pa kinsonian mice being mainly due o he elease o GDNF by he s ia al g a [ 19 ], we analyzed he amoun o he dopamino ophic ac o p oduced by a CB neu osphe es in compa ison wi h o he issues. GDNF was measu ed using a speci ic enzyme-linked immunoso ben assay (ELISA) in he a supe io ce ical ganglion (SCG), which does no exp ess GDNF [ 21 ], na i e CB o CB neu osphe es. As shown in Figu e 1c, he CB neu osphe es p oduced high le els o GDNF, simila o hose o he na i e CB issue. Mo eo e , we es ed whe he he abili y o p oduce GDNF by s em-cell-de i ed CB glomus cells was main ained a e an in as ia al ansplan a ion. Mouse CB neu osphe es cul u es we e pe o med om he e ozygous GDNF/lacZ animals (exp essing be a-galac osidase unde he con ol o GDNF p omo e ) and ansplan ed in o he s ia um o wild- ype mice (n= 5). In hese g a s, he cells exp essing GDNF can be labelled wi h cha ac e is ic X-gal s aining [ 19 , 21 ]. As indica ed in Figu e 1d, he in as ia al ansplan s o he GDNF/lacZ CB neu osphe es showed in ense X-gal s aining wi h nume ous cells exp essing GDNF. Rema kably, he combina ion o X-gal s aining wi h TH immuno luo escence e ealed a coincidence be ween X-gal deposi s and he a ea co e ed wi h TH + cells, indica ing ha s em-cell-de i ed dopamine gic CB glomus cells exp essed high le els o GDNF a e he s ia al ansplan a ion. O e all, hese da a clea ly showed ha in i o -expanded CB glomus cells p esen ed he same p ope ies (a ma u e dopamine gic pheno ype and a high GDNF exp ession e en a e an in ace eb al ansplan a ion) as na i e CB glomus cells. The e o e, ou indings suppo he use o CB-de i ed neu osphe es in an ipa kinsonian cell he apy. In . J. Mol. Sci. 2023, 24, 5575 4 o 13 Figu e 1. Cha ac e is ics o CB s em-cell-de i ed neu osphe es. (a,b) High-magni ica ion images o ou ep esen a i e examples o mouse (a) and a (b) CB s em-cell-de i ed neu osphe es a e TH ( ed), nes in (g een) and nuclea DAPI (blue) immuno luo escence. No e he p esence o blebs wi h abundan TH+ glomus cells in he a CB neu osphe es. (c) GDNF con en , exp essed in pg/μg o o al p o ein, o a supe io ce ical ganglion (n = 7; SCG), na i e CB issue (n = 6) and CB neu o- sphe es (n = 3). (d) GDNF exp ession in in as ia ally g a ed CB neu osphe es om GDNF/X-gal he e ozygous mice. The high magni ica ion images in he igh panels show he coincidence be- ween he GDNF/X-gal (blue) and TH ( ed) luo escence signals, clea ly demons a ing he GDNF exp ession on g a ed TH+ glomus cells. In c, he K uskal–Wallis es and pos hoc Dunn’s es we e pe o med. * p < 0.05; ** p < 0.01, in espec o SCG. 2.2. P o ec i e and Res o a i e E ec s o S em-Cell-De i ed CB Glomus Cell The apy on he Ni- g os ia al Pa hway The po en ial use o s em-cell-de i ed CB glomus cells in an ipa kinsonian cell he - apy was analyzed using in i o a CB-de i ed neu osphe es, which p o ide highe yields o di e en ia ed TH+ glomus cells han mouse CB neu osphe es (see Figu e 1a,b). Ra CB neu osphe es we e g a ed in ch onic MPTP pa kinsonian mice [19,37] subjec ed o an immunosupp essi e p o ocol de eloped by ou g oup ha allowed us o s udy he long- e m e ec s o neu al xenog a s [31]. In a i s assay, e med he “neu op o ec ion expe imen ”, we pe o med unila e al xenog a s o a CB neu osphe es, wi h a sham g a in he con ala e al hemisphe e as in e nal con ol, on ecep o mice ha we e sub- jec ed o immunosupp ession (see he Ma e ials and Me hods Sec ion and expe imen al scheme in Figu e 2a). Two weeks la e , xenog a ed mice we e ende ed pa kinsonian h ough he ch onic adminis a ion o MPTP (20 mg/Kg s.c.; h ee imes/week o 3 mon hs) and we e allowed o eco e om he oxic ea men o 1 mon h. A e wa ds, he CB xenog a ed mice we e eu hanized and a his ological examina ion o he SNpc TH+ neu ons was ca ied ou . In addi ion, o alida e he degene a ion o dopamine gic SNpc neu ons p oduced by he ch onic MPTP ea men , an expe imen al g oup o saline- ea ed mice was included. The his ological analysis o he s ia um e ealed i e animals wi h well-p ese ed in as ia al g a s and wi h abundan in i o-expanded di e en i- a ed TH+ dopamine gic CB glomus cells (Figu e 2b,c). A he le el o mesencephalon, he Figu e 1. Cha ac e is ics o CB s em-cell-de i ed neu osphe es. ( a , b ) High-magni ica ion images o ou ep esen a i e examples o mouse ( a ) and a ( b ) CB s em-cell-de i ed neu osphe es a e TH ( ed), nes in (g een) and nuclea DAPI (blue) immuno luo escence. No e he p esence o blebs wi h abundan TH + glomus cells in he a CB neu osphe es. ( c ) GDNF con en , exp essed in pg/ µ g o o al p o ein, o a supe io ce ical ganglion (n= 7; SCG), na i e CB issue (n= 6) and CB neu osphe es (n= 3). ( d ) GDNF exp ession in in as ia ally g a ed CB neu osphe es om GDNF/X- gal he e ozygous mice. The high magni ica ion images in he igh panels show he coincidence be ween he GDNF/X-gal (blue) and TH ( ed) luo escence signals, clea ly demons a ing he GDNF exp ession on g a ed TH + glomus cells. In c, he K uskal–Wallis es and pos hoc Dunn’s es we e pe o med. * p< 0.05; ** p< 0.01, in espec o SCG. In . J. Mol. Sci. 2023,24, 5575 4 o 12 2.2. P o ec i e and Res o a i e E ec s o S em-Cell-De i ed CB Glomus Cell The apy on he Nig os ia al Pa hway The po en ial use o s em-cell-de i ed CB glomus cells in an ipa kinsonian cell he apy was analyzed using in i o a CB-de i ed neu osphe es, which p o ide highe yields o di e en ia ed TH + glomus cells han mouse CB neu osphe es (see Figu e 1a,b). Ra CB neu osphe es we e g a ed in ch onic MPTP pa kinsonian mice [ 19 , 37 ] subjec ed o an immunosupp essi e p o ocol de eloped by ou g oup ha allowed us o s udy he long- e m e ec s o neu al xenog a s [ 31 ]. In a i s assay, e med he “neu op o ec ion expe imen ”, we pe o med unila e al xenog a s o a CB neu osphe es, wi h a sham g a in he con ala e al hemisphe e as in e nal con ol, on ecep o mice ha we e subjec ed o immunosupp ession (see he Ma e ials and Me hods Sec ion and expe imen al scheme in Figu e 2a). Two weeks la e , xenog a ed mice we e ende ed pa kinsonian h ough he ch onic adminis a ion o MPTP (20 mg/Kg s.c.; h ee imes/week o 3 mon hs) and we e allowed o eco e om he oxic ea men o 1 mon h. A e wa ds, he CB xenog a ed mice we e eu hanized and a his ological examina ion o he SNpc TH + neu ons was ca ied ou . In addi ion, o alida e he degene a ion o dopamine gic SNpc neu ons p oduced by he ch onic MPTP ea men , an expe imen al g oup o saline- ea ed mice was included. The his ological analysis o he s ia um e ealed i e animals wi h well- p ese ed in as ia al g a s and wi h abundan in i o -expanded di e en ia ed TH + dopamine gic CB glomus cells (Figu e 2b,c). A he le el o mesencephalon, he analysis o he SNpc o hese animals e ealed an impo an neu onal dea h o dopamine gic neu ons in he sham-g a ed side compa ed o he saline- ea ed mice (Table 1). Howe e , he ipsila e al SNpc o he CB neu osphe e g a showed a signi ican ly highe numbe o TH + nig al neu ons wi h espec o he con ala e al sham-g a ed hemisphe e (Table 1and Figu e 2b,d), indica ing he ophic p o ec ion induced by he g a ed CB glomus cells o e hese neu ons. Table 1. S e eological analysis o dopamine gic nig al neu ons and s ia al a icosi ies. Neu op o ec ion Expe imen nTH+SNpc Neu ons Saline Sham-g a ed MPTP CB-neu osphe es-g a ed MPTP 4 5 5 3148 ±81.45 * (p= 0.015) 1935 ±277.90 2496 ±330.50 * (p= 0.031) Res o a ion Expe imen nTH+S Va icosi ies (106/mm3) Saline Sham-g a ed MPTP CB-neu osphe es-g a ed MPTP 6 8 8 12.95 ±0.37 **** (p< 0.0001) 7.37 ±0.25. 11.14 ±0.48 **** (p< 0.0001) *p< 0.05 (saline s. sham-g a ed MPTP, Mann–Whi ney es ; sham-g a ed MPTP s. CB-neu osphe es-g a ed MPTP, pai ed Wilcoxon es ). **** p< 0.0001 (saline s. sham-g a ed MPTP, unpai ed - es ; sham-g a ed MPTP s. CB-neu osphe es-g a ed MPTP, pai ed - es ). We also s udied whe he he ansplan a ion o CB neu osphe es could exe neu- o egene a i e ac ions (e.g., axonal sp ou ing) on a p e iously damaged dopamine gic s ia al inne a ion. In his assay, e med he “ es o a ion expe imen ”, ecep o mice we e subjec ed o he ch onic MPTP ea men o h ee mon hs, and once he oxic ea - men was inished and he i e e sible damage on nig os ia al dopamine gic neu ons induced [ 19 , 37 ], xenog a s wi h a CB neu osphe es we e pe o med. Xenog a ed CB mice we e immunosupp essed a e he ansplan a ion and eu hanized ou weeks la e o he his ological examina ion (see expe imen al scheme in Figu e 3a). All mice ana- lyzed unde his expe imen al design (n= 8) e ealed well-main ained xenog a s wi h nume ous dopamine gic CB glomus cells (Figu e 3b,c). As expec ed, in he sham-g a ed s ia um he ch onic MPTP ea men induced an impo an dec ease in dopamine gic inne a ion, e ealed wi h measu emen s o he TH + op ical densi y (O.D.; saline, 19.69 ± 1.11 a bi a y uni s (a.u.) s. he sham-g a ed MPTP, 11.25 ± 0.71 a.u.; p< 0.0001) o h ough he s e eological analysis o TH + s ia al a icosi ies (Table 1). Rema kably, In . J. Mol. Sci. 2023,24, 5575 5 o 12 he s ia al xenog a s o he in i o -expanded CB glomus cells p oduced a signi ican inc ease in he dopamine gic s ia al inne a ion in compa ison wi h he con ala e al sham-g a ed hemisphe e (Table 1and Figu e 3b,d,e). These esul s demons a ed ha he s ia al ansplan a ion o CB neu osphe es can induce an ou g ow h (sp ou ing) o dopamine gic s ia al ibe s. Taken oge he , he esul s ob ained bo h in he neu op o ec- ion and es o a ion expe imen s clea ly indica ed ha s em-cell-de i ed CB glomus cells main ained a simila neu o ophic po en ial as na i e CB issue, inducing ophic p o ec ion and he es o a ion o he nig os ia al pa hway a e he s ia al ansplan a ion. In . J. Mol. Sci. 2023, 24, 5575 5 o 13 analysis o he SNpc o hese animals e ealed an impo an neu onal dea h o dopamin- e gic neu ons in he sham-g a ed side compa ed o he saline- ea ed mice (Table 1). Howe e , he ipsila e al SNpc o he CB neu osphe e g a showed a signi ican ly highe numbe o TH+ nig al neu ons wi h espec o he con ala e al sham-g a ed hemisphe e (Table 1 and Figu e 2b,d), indica ing he ophic p o ec ion induced by he g a ed CB glomus cells o e hese neu ons. Table 1. S e eological analysis o dopamine gic nig al neu ons and s ia al a icosi ies. Neu op o ec ion Expe imen n TH+ SNpc Neu ons Saline Sham-g a ed MPTP CB-neu osphe es-g a ed MPTP 4 5 5 3148 ± 81.45 * (p = 0.015) 1935 ± 277.90 2496 ± 330.50 * (p = 0.031) Res o a ion Expe imen n TH+ S Va icosi ies (106/mm3) Saline Sham-g a ed MPTP CB-neu osphe es-g a ed MPTP 6 8 8 12.95 ± 0.37 **** (p < 0.0001) 7.37 ± 0.25. 11.14 ± 0.48 **** (p < 0.0001) * p < 0.05 (saline s. sham-g a ed MPTP, Mann–Whi ney es ; sham-g a ed MPTP s. CB-neu o- sphe es-g a ed MPTP, pai ed Wilcoxon es ). **** p < 0.0001 (saline s. sham-g a ed MPTP, un- pai ed - es ; sham-g a ed MPTP s. CB-neu osphe es-g a ed MPTP, pai ed - es ). Figu e 2. P o ec ion o dopamine gic SNpc neu ons h ough s ia al xenog a s o CB s em-cell-de- i ed glomus cells. (a) Scheme o he expe imen al p o ocol used o analyze he neu op o ec i e e ec o in i o-expanded a CB cells on mice dopamine gic nig os ia al neu ons. (b) B ain co o- nal sec ions, a e TH immunohis ochemis y, showing he sham (le panels) and CB-neu osphe es- g a ed ( igh panels) hemisphe es a he le el o s ia um ( op) and SNpc (middle and bo om). (c) Image, a highe magni ica ion and a e TH immunos aining, o he CB s em-cell-de i ed cells g a ed in he s ia um, showing an abundan numbe o in i o-expanded dopamine gic glomus cells. (d) S e eological quan i ica ion, exp essed as pe cen age o sham-g a ed side, o ipsila e al TH+ SNpc neu ons o he g a ed s ia um. Pai ed Wilcoxon es ; n = 5; * p < 0.05. Figu e 2. P o ec ion o dopamine gic SNpc neu ons h ough s ia al xenog a s o CB s em-cell- de i ed glomus cells. ( a ) Scheme o he expe imen al p o ocol used o analyze he neu op o ec i e e ec o in i o -expanded a CB cells on mice dopamine gic nig os ia al neu ons. ( b ) B ain co onal sec ions, a e TH immunohis ochemis y, showing he sham (le panels) and CB-neu osphe es- g a ed ( igh panels) hemisphe es a he le el o s ia um ( op) and SNpc (middle and bo om). ( c ) Image, a highe magni ica ion and a e TH immunos aining, o he CB s em-cell-de i ed cells g a ed in he s ia um, showing an abundan numbe o in i o -expanded dopamine gic glomus cells. ( d ) S e eological quan i ica ion, exp essed as pe cen age o sham-g a ed side, o ipsila e al TH + SNpc neu ons o he g a ed s ia um. Pai ed Wilcoxon es ; n= 5; * p< 0.05. In . J. Mol. Sci. 2023,24, 5575 6 o 12 In . J. Mol. Sci. 2023, 24, 5575 6 o 13 We also s udied whe he he ansplan a ion o CB neu osphe es could exe neu o- egene a i e ac ions (e.g., axonal sp ou ing) on a p e iously damaged dopamine gic s i- a al inne a ion. In his assay, e med he “ es o a ion expe imen ”, ecep o mice we e subjec ed o he ch onic MPTP ea men o h ee mon hs, and once he oxic ea men was inished and he i e e sible damage on nig os ia al dopamine gic neu ons induced [19,37], xenog a s wi h a CB neu osphe es we e pe o med. Xenog a ed CB mice we e immunosupp essed a e he ansplan a ion and eu hanized ou weeks la e o he his- ological examina ion (see expe imen al scheme in Figu e 3a). All mice analyzed unde his expe imen al design (n = 8) e ealed well-main ained xenog a s wi h nume ous do- pamine gic CB glomus cells (Figu e 3b,c). As expec ed, in he sham-g a ed s ia um he ch onic MPTP ea men induced an impo an dec ease in dopamine gic inne a ion, e- ealed wi h measu emen s o he TH+ op ical densi y (O.D.; saline, 19.69 ± 1.11 a bi a y uni s (a.u.) s. he sham-g a ed MPTP, 11.25 ± 0.71 a.u.; p < 0.0001) o h ough he s e eo- logical analysis o TH+ s ia al a icosi ies (Table 1). Rema kably, he s ia al xenog a s o he in i o-expanded CB glomus cells p oduced a signi ican inc ease in he dopamine - gic s ia al inne a ion in compa ison wi h he con ala e al sham-g a ed hemisphe e (Ta- ble 1 and Figu e 3b,d,e). These esul s demons a ed ha he s ia al ansplan a ion o CB neu osphe es can induce an ou g ow h (sp ou ing) o dopamine gic s ia al ibe s. Taken oge he , he esul s ob ained bo h in he neu op o ec ion and es o a ion expe imen s clea ly indica ed ha s em-cell-de i ed CB glomus cells main ained a simila neu- o ophic po en ial as na i e CB issue, inducing ophic p o ec ion and he es o a ion o he nig os ia al pa hway a e he s ia al ansplan a ion. Figu e 3. Res o a i e e ec s o CB neu osphe es xenog a s on dopamine gic s ia al ibe s. (a) Scheme o he expe imen al p o ocol used o es he epa a i e e ec o in i o-expanded a CB cells on mouse dopamine gic s ia al e minals. (b) B ain co onal sec ions, a e TH immunohis o- chemis y, o he sham- (le ) and CB-neu osphe es-g a ed ( igh ) s ia a (uppe panels). Inse s de- pic ed in lowe panels illus a e he epai h ough axonal sp ou ing o he dopamine gic s ia al e minals induced by he implan . (c) Image, a highe magni ica ion and a e TH immunos aining, showing he g a o CB s em-cell-de i ed TH+ glomus cells wi h abundan numbe o in i o-ex- panded dopamine gic glomus cells. (d) Analysis o s ia al dopamine gic inne a ion h ough meas- u emen s o TH+ op ical densi y (O.D.). (e) S e eological quan i ica ion o dopamine gic s ia al Figu e 3. Res o a i e e ec s o CB neu osphe es xenog a s on dopamine gic s ia al ibe s. ( a ) Scheme o he expe imen al p o ocol used o es he epa a i e e ec o in i o -expanded a CB cells on mouse dopamine gic s ia al e minals. ( b ) B ain co onal sec ions, a e TH immunohis ochemis y, o he sham- (le ) and CB-neu osphe es-g a ed ( igh ) s ia a (uppe panels). Inse s depic ed in lowe panels illus a e he epai h ough axonal sp ou ing o he dopamine gic s ia al e minals induced by he implan . ( c ) Image, a highe magni ica ion and a e TH immunos aining, showing he g a o CB s em-cell-de i ed TH + glomus cells wi h abundan numbe o in i o -expanded dopamine gic glomus cells. ( d ) Analysis o s ia al dopamine gic inne a ion h ough measu emen s o TH + op ical densi y (O.D.). ( e ) S e eological quan i ica ion o dopamine gic s ia al a icosi ies. In (d,e), da a a e exp essed as pe cen age o sham-g a ed side. Pai ed Wilcoxon es ; n= 8; ** p< 0.01. 2.3. Su i al o S em-Cell-De i ed CB Glomus Cell In as ia al T ansplan s A cha ac e is ic o CB glomus cells o ele ance o neu al ansplan a ion is hei ex ao dina y long su i al in ce eb al implan s. This ac has been a ibu ed o he physiological esis ance o CB glomus cells o hypoxia [ 18 , 20 , 26 , 30 ], a condi ion p esen in in ace eb al g a s [ 38 ]. In his wo k, we s udied he su i al o he xenog a s o in i o -gene a ed TH + CB glomus cells a e an in as ia al ansplan a ion. In he i s analysis, xenog a s o CB neu osphe es we e pe o med in immunosupp essed mice ha we e no ea ed wi h MPTP, and we e eu hanized o he his ological examina ion (n= 7) a di e en imepoin s (be ween 2 and 12 weeks). In his analysis, all he g a s emained ali e wi h nume ous TH + glomus cells, showing a simila su i al as p e iously desc ibed o xeno ansplan s o na i e CB issue [ 31 ]. In he case o he neu op o ec ion expe imen (see Figu e 2a), in which he CB neu osphe es xenog a s we e pe o med be o e he ch onic MPTP ea men , only 6 xenog a s (1 loca ed in he co ex and 5 loca ed in he s ia um) o he 32 g a ed mice su i ed (~20% su i al). In con as , in he es o a ion expe imen (see Figu e 3a), whe e he ansplan s we e ca ied ou once he ch onic MPTP ea men was inished and he dopamine gic oxin was washed ou , 9 xenog a s (1 loca ed in he co ex and 8 in he s ia um) o 11 g a ed mice emained ali e (~82% su i al). The low su i al o he CB neu osphe es xeno ansplan s obse ed in he neu op o ec ion expe imen seemed o be ela ed o a oxic e ec o he MPTP ea men on he g a ed in i o -expanded TH + glomus cells, because high su i al a es we e obse ed in he o he In . J. Mol. Sci. 2023,24, 5575 7 o 12 wo expe imen s, in which g a ed cells we e no exposed o he neu o oxin. Mo eo e , he six xenog a s ha su i ed in he neu op o ec ion expe imen had abundan and appa en ly heal hy TH + CB cells, e en i e mon hs a e ansplan a ion, sugges ing ha no all in i o cul u es o CB neu osphe es had he same deg ee o suscep ibili y o he dopamine gic oxin. 3. Discussion CB cell he apy has been p o en o be e ec i e in oden and nonhuman p ima e p eclinical PD models [ 16 – 19 , 31 , 39 – 41 ]. The analyses o he mechanism unde lying he an ipa kinsonian ac ion o CB ansplan s e ealed ha hei bene icial e ec was due o a ophic s imula ion o he nig os ia al pa hway, mainly media ed by GDNF eleased by g a ed CB glomus cells [ 18 , 19 , 21 ]. Fu he mo e, wo pilo phase I/II open ials e alua ed he easibili y, sa e y and clinical e icacy o CB au o ansplan a ion in PD pa ien s [ 23 , 24 ], showing ha i is a sa e and easible p ocedu e ha p oduces a clinical imp o emen in mode a ely a ec ed pa ien s, simila o ha ob ained a e h VM g a s [ 6 , 7 ]. Howe e , he e ec i eness o CB ansplan a ion obse ed in PD pa ien s was shown o be conside ably lowe han in expe imen al models. Among he ac o s ha could in luence he clinical ou come o CB cell he apy, he sca ci y o he g a ed issue appea s as a p incipal one [ 24 ]. In his wo k, we e alua ed he po en ial use o in i o -gene a ed dopamine gic CB glomus cells h ough he cul u e and di e en ia ion o neu al s em cells p esen in he o gan, as a way o inc ease he amoun o issue a ailable o g a ing in PD pa ien s. P e ious epo s demons a ed ha s em-cell-de i ed CB glomus cells ha e a highly di e en ia ed dopamine gic pheno ype, exp essing ol age-ga ed Ca 2+ and K + channels and sec e ing ca echolamines in esponse o hypoxia o hypoglycemia as ma u e glomus cells [ 27 , 28 , 42 ]. He e, we also showed ha in i o -gene a ed TH + glomus cells ha e simila neu o ophic ea u es o na i e CB glomus cells, p oducing compa able amoun s o GDNF and main ain- ing he exp ession o he neu o ophic ac o a e a s ia al ansplan a ion. The gene a ion o di e en ia ed dopamine gic CB glomus cells h ough he in i o cul u e o CB neu al s em cells has been desc ibed in a s, mice and humans [ 27 – 29 , 43 ]. Howe e , ou s udy e- ealed impo an a ia ions in e ms o p oli e a ion and di e en ia ion in TH + CB glomus cells be ween a s and mice, wi h a neu osphe es being much la ge and ha ing mo e dopamine gic cells han mouse neu osphe es. Addi ional expe imen al wo k should be pe o med o iden i y he mechanisms o he p oli e a ion and di e en ia ion o CB s em cells, in o de o inc ease he yield o in i o -expanded CB dopamine gic glomus cells o hei use in an ipa kinsonian cell he apy. The analysis o he su i al o s em-cell-de i ed CB glomus cell in ace eb al implan s e ealed wo ele an indings. On he one hand, s ia al xenog a s o in i o -gene a ed TH + CB glomus cells showed simila a es o su i al (~80–100%) as p e iously epo ed o bo h xeno- and allo ansplan a ions o na i e CB cells [ 18 , 19 , 31 ]. On he o he hand, ce eb al implan s o in i o -gene a ed CB glomus cells p esen ed an ele a ed suscep ibili y o he dopamine gic neu o oxin MPTP, an obse a ion ha con as s wi h he esis ance epo ed o na i e CB issue [ 19 , 21 , 31 ]. The ac o s ha de e mine his high suscep ibili y o s em- cell-de i ed CB glomus cells o MPTP a e unknown, and should be add essed in u u e expe imen al s udies. I is also ele an o no e ha we did no see signs o umo igenici y o o he dele e ious e ec s in any o he g a ed mice a he di e en imepoin s s udied (2, 4, 5, 12 and 18 weeks), indica ing he sa e y o he in ace eb al g a ing o in i o -gene a ed dopamine gic CB glomus cells. To s udy whe he he ansplan s o s em-cell-de i ed CB glomus cells could induce neu o ophic p o ec ion and es o a ion (axonal sp ou ing) o damaged dopamine gic nig al neu ons, we used a me hodology p e iously alida ed o s ia al g a s o na i e CB cells [ 19 , 31 ]. Rema kably, he neu op o ec i e and es o a i e e ec s exe ed by he xenog a s o in i o -gene a ed dopamine gic CB cells we e simila o hose p e iously desc ibed o allog a s [ 19 ] o xenog a s [ 31 ] o na i e CB cells in he ch onic MPTP pa kinsonian model. The ac ha he immunosupp essi e p o ocol used did no al e he In . J. Mol. Sci. 2023,24, 5575 8 o 12 an ipa kinsonian e ec s o s em-cell-de i ed CB glomus cells opens he possibili y o ob ain in i o -gene a ed human CB glomus cells no only om PD pa ien s, h ough he unila e al esec ion o he o gan, bu also om allogenic dono s. In conclusion, he da a p esen ed he e clea ly showed ha s em-cell-de i ed CB glomus cells ha e simila neu o ophic ea u es o na i e issue. S em-cell-de i ed dopamine gic and GDNF-p oducing glomus cells ep esen a new he apeu ic op ion o be conside ed in cell he apy in PD. These cells could be use ul ei he o inducing neu o ophic p o ec ion and he s imula ion o damaged SNpc neu ons o o imp o ing he su i al and e icacy o h VM ansplan s [ 9 , 44 , 45 ]. The neu o ophic po en ial o s em-cell-de i ed CB glomus cells could also be ele an o he ea men o o he neu odegene a i e diseases [46,47]. 4. Ma e ials and Me hods 4.1. Animal Ca e and Pha macological T ea men s C57BL/6N and he e ozygous GDNF/lacZ mice o 2-3 mon hs o age (CEA-Osca Pin ado, Uni e si y o Se ille) we e housed in a empe a u e- egula ed en i onmen (22 ±1◦C ) on a 12 h ligh /da k cycle wi h ad libi um access o ood and wa e . The mice we e ende ed pa kinsonian as p e iously desc ibed [ 19 , 31 , 37 ] h ough he subcu a- neous (s.c.) adminis a ion o MPTP (20 mg/kg; Sigma, S . Louis, MO, USA) 3 imes pe week o 3 mon hs. An immunosupp essi e ea men was applied o he xenog a ed mice as p e iously indica ed [ 31 ]. B ie ly, animals ecei ed daily injec ions o cyclospo ine A (CsA; 15 mg/Kg; Sigma; s.c.) and p ednisone (P ed; 20 mg/Kg; Sigma; s.c.) du ing he i s wo weeks, al e na ing daily injec ions o CsA (15 mg/Kg; s.c.) o P ed (20 mg/Kg; s.c.) o 1 week and 3 doses pe week o CsA (15 mg/Kg; s.c.) un il hey we e eu hanized o he his ological analysis. A he end o he expe imen s, he mice we e eu hanized unde deep anes hesia induced wi h a combina ion o 100 mg/kg ke amine (P ize , New Yo k, NY, USA) and 10 mg/kg xylazine (Baye , Le e kusen, Ge many). All expe imen s we e ca ied ou acco ding o he Eu opean Di ec i e 2010/63/EU and he Spanish RD/53/2013 o he p o ec ion o animals used o scien i ic pu poses. The animal p ocedu es we e app o ed by he Animal Resea ch Commi ee o he Uni e si y Hospi al Vi gen del Rocío (Uni e si y o Se ille; egis e ed ag eemen numbe 11-07-14-112). 4.2. CB Neu osphe e Cul u es Ra and mouse CB cell dissocia ion and neu osphe es assays we e ca ied ou as de- sc ibed by ou labo a o y [ 27 , 28 , 35 , 48 ]. CBs we e dissocia ed using an enzyma ic ea men wi h 0.6 mg/mL collagenase ype II (Sigma, S . Louis, MO, USA), 0.3 mg/mL ypsin (Sigma) and 5 mg/mL po cine panc eas elas ase (Calbiochem, San Diego, CA, USA) in a PBS solu ion and 50 µ M CaCl 2 o 20 min a 37 ◦ C in a c ys al lask a 600 pm in a The momixe Com o (Eppendo , Hambu g, Ge many). A e ha , 2 olumes o blocking solu ion ( o 50 mL: 44 mL L15 medium (GIBCO, Wal ham, MA, USA), 0.5 mL penicillin/s ep omycin (GIBCO), 0.5 mL 1 M HEPES bu e (GIBCO), 2.5 mL FBS 5% (GIBCO) and 2.5 mL dis illed and deionized wa e ) we e added o quench he enzymes. Dissocia ed CB cells we e cen- i uged o 5 min a 300 × ga 4 ◦ C and he cell pelle was esuspended and pla ed on ul alow binding 6-well pla es (Co ning, New Yo k, NY, USA) a a clonal densi y o 10.000 cells pe well. The cul u e medium con ained D-MEM:F-12 (GIBCO) wi h 15% FBS (GIBCO), 1% N2 supplemen (GIBCO), 2% B27 supplemen (GIBCO), 1% penicillin/s ep omycin (GIBCO), 20 ng/mL ecombinan human bFGF (R&D Sys ems, Minneapolis, MN, USA), 20 ng/mL ecombinan human IGF-1 (R&D Sys ems) and 20 ng/mL ecombinan human EGF (R&D Sys ems). All cul u es we e main ained in O 2 - and CO 2 -con olled incuba o s (The mo, Wal ham, MA, USA) a 3% and 5% espec i ely and 37 ◦C o 10–12 days be o e being used o g a ing. 4.3. CB Neu osphe e Xenog a ing Mice g a ed wi h a CB neu osphe es xenog a s ecei ed a CsA injec ion (15 mg/kg; s.c.) 4–6 h be o e s a ing he su gical p ocedu e. An in as ia al g a o CB neu osphe es In . J. Mol. Sci. 2023,24, 5575 9 o 12 was pe o med as p e iously desc ibed o na i e CB issue [ 19 , 31 ]. B ie ly, 100–150 CB neu osphe es we e placed in 1 µ L o Ty ode’s solu ion (140 mM NaCl; 4.7 mM KCl; 2 mM CaCl 2 ; 1 mM MgCl 2 ; 10 mM 4-2-hyd oxye hyl-1-pipe azinee hanesul onic acid, HEPES; Sigma; in dis illed and deionized wa e ) and we e s e eo axically injec ed in o he s ia um ( om b egma in mm: an e opos e io , +0.4; la e al, +2; en al, − 3.5) wi h a 25-gauge sy inge (Hamil on, Bonaduz, Ge many) acco ding o he mouse b ain s e eo axic a las [ 49 ]. As an in e nal con ol, a sham g a was pe o med in he con ala e al s ia um, injec ing 1 µ L o he ehicle solu ion. To a oid di e ences be ween animals, s e eologic and densi ome ic alues (see below) o CB-neu osphe e-g a ed pa kinsonian mice we e exp essed as a pe cen age o he sham-g a ed hemisphe e. 4.4. His ological Analyses Mice we e ansca dially pe used wi h 50 mL o PBS (Sigma) and 50 mL o 4% pa a o maldehyde (Sigma) in PBS. The b ains we e immedia ely emo ed and ixed o e nigh a 4 ◦ C wi h 4% pa a o maldehyde in PBS. A e ixa ion, he b ains we e c yop o ec ed in 30% suc ose (Sigma) in PBS and included in an op imum cu ing em- pe a u e compound (O.C.T. compound, Tissue-Tek, Tokyo, Japan). The neu osphe es we e ixed in 4% pa a o maldehyde in PBS o 20 min, washed 3 imes in PBS o 5 min each, c yop o ec ed in 30% suc ose o 30 min and, inally, embedded in he OCT com- pound and ozen. B ain co onal sec ions (30 µ m hickness) o embedded neu osphe es (10 µm hickness) we e cu on a c yos a (Leica, We zla , Ge many). Ty osine hyd oxylase (TH) and nes in immunode ec ion was pe o med as p e iously desc ibed [ 19 , 31 , 48 ] us- ing, espec i ely, polyclonal an i-TH (1:1000; No us Biologicals, NB300-109), monoclonal an i-nes in (1:500; Me ck-Millipo e, Bu ling on, MA, USA, MAB353) and a seconda y pe oxidase-conjuga ed an ibody ki (Dako, Ho eds aden, Denma k) o luo escence sec- onda y an ibodies (goa –an i abbi Alexa568, 1:400, In i ogen; goa –an imouse Alexa488, 1:400, Jackson ImmunoResea ch, Wes G o e, PA, USA). The image acquisi ion and analysis we e pe o med wi h ligh ansmi ed (AX70 o Bx61, bo h wi h digi al e ige a ed came a DP72, Olympus, Tokyo, Japan) and hei speci ic imaging so wa e (CellSens 1.4.1). 4.5. GDNF ELISA The GDNF p o ein con en was measu ed using he me hodology desc ibed be o e by ou g oup [ 21 , 44 ]. B ie ly, a SCG, CB and CB neu osphe es we e ozen in liquid N 2 and homogenized in a lysis bu e (137 mM NaCl; 20 mM T is, pH 8.0; 1% IGEPAL CA-630, 10% glyce ol, 1/1000 p o ease inhibi o cock ail; Sigma) using a poly on (OMNI, Wa e bu y, CT, USA). P o ein ex ac ion and he ELISA assay we e pe o med ollowing he manu ac u e s’ ins uc ions (G7621, P omega, Madison, WI, USA), excep o he an i-GDNF monoclonal and an i-hGDNF polyclonal an ibodies, which we e used a 1:500 and 1:250, espec i ely. The abso bance a 450 nm was measu ed in a pla e eade (The mo) and he o al p o ein con en o he samples was ob ained using he B ad o d assay (Bio-Rad, He cules, SA, USA). 4.6. S e eology and Densi ome y Unbiased s e eological analyses we e pe o med wi h he use o sys ema ic andom sampling using he op ical dissec o me hod [ 50 ]. All s e eological p ocedu es we e pe - o med using he C.A.S.T. g id sys em (Olympus) o new CAST TM sys em (Visiopha m, Hø sholm, Denma k), ollowing he me hodology desc ibed by ou labo a o y [ 19 , 31 , 37 ]. Es ima ions o TH + SNpc neu ons and s ia al dopamine gic a icosi ies we e ca ied ou in he egions spanning om − 3.08 mm o − 3.28 mm (SNpc) and om 0.80 mm o 0.10 mm (s ia um), ela i e o B egma, acco ding o he F anklin and Paxinos mouse b ain s e eo axic a las [ 49 ]. Only SNpc cells la e al o he medial e minal nucleus o he accesso y op ic ac we e de e mined o ha e a clea sepa a ion om he adjacen en al egmen al a ea [ 51 ]. Re e ence olumes o each sec ion we e ou lined unde low magni ica ion (4 × ), and TH + neu ons o a icosi ies we e coun ed a high magni ica ion (40 × ) using,