Neural development by transplanted human embryonal carcinoma stem cells expressing green fluorescent protein

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.     

绿色荧光蛋白标记神经干细胞的体外研究

目的　构建携带绿色荧光蛋白 (GFP)基因的逆转录病毒载体pLNCX2 GFP ,用GFP对神经干细胞进行标记示踪。方法　应用基因克隆的方法 ,制备 pLNCX2 GFP ,借助阳离子脂质体转染包装细胞PA3 17,G418筛选阳性克隆 ,获取病毒上清 ;从胚胎大鼠脑中解剖分离和培养神经干细胞 ,用病毒上清感染大鼠胚胎神经干细胞。结果　经酶切电泳和DNA测序表明成功构建了重组GFP逆转录病毒 ,pLNCX2 GFP转染包装细胞后可以产生GFP逆转录病毒 ,病毒感染大鼠胚胎神经干细胞可以长期表达绿色荧光。结论　逆转录病毒能够快速、稳定、长期地将GFP基因转入神经干细胞 ,这种标记方法非常有利于神经干细胞移植后结构和功能的研究     

不同转移特性稳定表达绿色荧光蛋白的乳腺癌MCF7细胞亚株的建立

目的 建立不同转移特性并稳定表达绿色荧光蛋白的人乳腺癌MCF7细胞亚株.方法 转染绿色荧光蛋白(GFP)入MCF7细胞,通过有限稀释法得到10株亚株,细胞电泳得到电泳率最高(A2)、最低(C6)的两株,测定其增殖率、克隆形成率、体外侵袭能力,行裸鼠皮下、原位成瘤试验.结果 获得稳定表达绿色荧光蛋白不同转移特性的细胞亚株A2和C6,A2的群体倍增时间为25.6 h,C6为41.8 h;软琼脂形成率A2为30.7%,C6为14%;体外侵袭能力,A2的平均侵袭细胞数为(136.80±12.36)个,明显高于C6(73.62±9.76)个;裸小鼠皮下成瘤试验发现A2成瘤力强,并且A2原位接种后可出现骨转移.结论 建立的细胞亚株有不同转移特性,有助于进一步研究乳腺癌骨转移的分子机制和治疗.     

表达增强型绿色荧光蛋白的骨肉瘤细胞亚株建立及生物学特性

目的 建立经绿色荧光蛋白基因转染的骨肉瘤细胞亚株并研究其生物特性。方法 利用脂质体转染增强型绿色荧光蛋白真核表达质粒（pEGFP-N1）人人骨肉瘤MG63细胞系，通过有限稀释法和细胞电泳，获得两株细胞克隆M6和M8，经体外细胞增殖、软琼脂形成、生长曲线、裸鼠成瘤试验综合分析其生物学行为改变。应用组织形态学观察、染色体分析、软琼脂克隆形成法研究癌细胞的生物学特性。结果 M6和M8两株在细胞电泳率和侵袭性上差异有统计学意义（P〈0．05），其中M6的群体倍增时间为38．4h，软琼脂形成率为18．7％，M8群体倍增时间为23．0h，软琼脂形成率为29．3％。裸小鼠背部皮下接种M6和M8，发现M8成瘤时间短，细胞增殖快，但在4周内两者均不发生转移。结论 骨肉瘤细胞亚系有不同的转移特性，GFP的整合及表达未对MG63细胞的生长状态造成明显影响，可作为报告基因进一步了解骨肉瘤细胞转移的差异性分析。     

大鼠胚胎肝干细胞的分离、鉴定及转染绿色荧光蛋白基因的研究

目的 分离、培养大鼠胚胎肝干细胞,检测肝干细胞表面标志CD49f,c-Met,建立携带示踪标记的大鼠肝干细胞.方法 Percoll梯度离心法分离孕14 d大鼠胚胎肝细胞,体外培养后采用流式细胞术(FACS)检测CIM9f和c-Met,pAcGFP1-N1质粒提取并经电泳鉴定后用脂质体法转染细胞.结果 成功分离并纯化了孕14 d大鼠胚胎肝细胞;CIM9f和c-Met阳性细胞的比例分别为65.0%和85.9%;电泳鉴定质粒正确,成功转染细胞后荧光显微镜观察到大量表达绿色荧光蛋白(GFP)的肝干细胞.结论 从孕14 d大鼠胎肝中成功分离并鉴定肝干细胞,成功转染pAeGFP1-N1质粒,建立携带稳定表达GFP示踪标记的大鼠肝干细胞.     

绿色荧光蛋白基因逆转录病毒载体转染人骨髓间充质干细胞及表达的研究

目的　绿色荧光蛋白 (EGFP)基因的逆转录病毒载体转染人骨髓间充质干细胞(MSCs)及其表达情况。方法　pEGFP与逆转录病毒载体经过酶切、连接等构建重组的EGFP 逆转录病毒载体 ,转染PA3 17细胞 ,用G418筛选出抗性克隆 ,获病毒上清 (滴度达到 8.5× 10 5cfu/ml)并感染人MSCs。流式细胞仪测定转染效率后加入成骨细胞分化培养夜 (地塞米松 (10 -7mol/L)、β 甘油磷酸钠 (10mmol/L)、维生素C(5 0mg/L ) ) ,2周后观察转染细胞的分化情况。 结果　 48h后细胞转染效率为 7% ,G418筛选 2周后约 97%细胞出现抗性克隆 ,表达维持 4周。基因转染细胞能够分化为成骨细胞。结论　重组EGFP逆转录病毒载体能转染MSCs ,且不影响细胞的功能。     

Dynamics of medial smooth muscle changes after carotid artery transplantation in transgenic mice expressing green fluorescent protein

BACKGROUND: Recent observations have demonstrated the importance of host cells in neointima formation after transplantation. Because little is known regarding the dynamics of host-derived cells in the graft media, we investigated this question in a mouse carotid artery transplantation model. METHODS: C57BL/6 carotid arteries were orthotopically transplanted into BALB/c mice ubiquitously expressing enhanced green fluorescent protein. Grafts were harvested at 1, 2, 4, and 8 weeks after transplantation for histologic examination. No immunosuppression was used. RESULTS: Immunostaining and semiquantitative analysis of cross sections showed that donor medial smooth muscle cells decreased over time in the graft media, whereas green fluorescent protein-positive/smooth muscle alpha-actin-positive cells (i.e., cells of host origin) increased over time. Interestingly, host cells were located only in the inner media and the neointima at 2 weeks and thereafter also in the outer media, indicating that the host-derived cells entered the media from the luminal side rather than from the adventitia. In longitudinal sections, there were no differences in the accumulation of donor- and host-derived cells between the end and middle regions of the graft media at 8 weeks. CONCLUSIONS: After transplantation, medial cells were replaced by alpha-actin-expressing host cells that were probably derived from circulating precursor cells. Our observations differ from the traditional view of a major contribution of donor medial smooth muscle cells to the neointima formation. Thus, circulating progenitor cells may be important for graft vessel disease.     

腺病毒介导神经生长因子在绿色荧光蛋白基因标记的间充质干细胞中表达及意义

目的为提高间充质干细胞(MSCs)表达神经生长因子(NGF)的能力,同时使MSCs 被绿色荧光蛋白(GFP)稳定标记,探讨通过病毒载体将NGF和GFP基因高效转染到MSCs的方法。方法取2个月龄100～150 g Wistar大鼠,全骨髓法分离培养纯化大鼠MSCs,利用携带人 NGFβ基因的复制缺陷性重组腺病毒(Ad-hNGFβ)和携带GFP基因的复制缺陷性逆转录病毒(Rt- GFP)转染MSCs,荧光显微镜观察GFP阳性的MSCs,免疫细胞化学和Western blot检测hNGFβ的表达。结果 Rt-GFP转染后,80％～90％MSCs可激发出明亮的绿色荧光,且荧光不随培养时间延长和传代次数增加而衰减。Ad-hNGFβ的转染MSCs中hNGFβ阳性率(90．17±2．14)显著高于阴性对照组(2．17±0．75,P<0．01)和空白对照组(1．83±0．98,P<0．01),转染后MSCs中NGFβ表达量(188．67±8．71)显著高于阴性对照组(25．67±4．08,P<0．01)和空白对照组(27．50± 3．33,P<0．01)。结论 Ad-hNGFβ可以高效转染MSCs,实现NGF在MSCs中高效表达,Rt-GFP 可以使MSCs获得长效标记。     

增强型绿色荧光蛋白基因转染小鼠骨髓间充质干细胞的研究

目的：以重组腺病毒（Ad）为载体,将增强型绿色荧光蛋白（EGFP）基因转染至小鼠骨髓间充质干细胞（mMSCs）中,为MSCs体内示踪提供实验基础。方法：用全骨髓细胞贴壁法分离纯化小鼠MSCs并体外扩增、鉴定,以重组绿色荧光蛋白基因的腺病毒（Ad-EGFP）转染,并用荧光显微镜和流式细胞仪检测转染率,CCK-8法检测转染细胞的增殖活性。结果：转染后10h MSCs开始表达荧光,6～8天达高峰,转染率可达92.3%,28天仍有表达;转染细胞的增殖活性和未转染细胞无统计学差异。结论：Ad-EGFP能高效、安全的转染mMSCs。     

胚胎干细胞分化为软骨细胞的研究现状与展望

胚胎干细胞具有多向分化潜能,在组织修复治疗方面具有广阔的应用前景.近年研究表明,胚胎干细胞在细胞因子、激素、微环境等作用下能够分化为软骨细胞.本文对近年来胚胎干细胞向软骨细胞定向分化中的各种因素进行综述.     

Successful generation of donor specific hematopoietic stem cell lines from co-cultured bone marrow with human embryonic stem cell line: a new methodology

We report the generation of 30 healthy human embryonic stem cell (h-ESC) lines from 33 voluntary oocyte donors using a donor somatic cell nuclear transfer (SCNT) technique on 190 oocytes. Our aim was to coculture them with their own bone marrow (BM) to generate hematopoietic progenitor cells for therapeutic purposes. Pluripotency and undifferentiated stage were confirmed using molecular cell surface markers. Normal karyotype of these cell lines was confirmed. Here we demonstrate that SCNT-h-ESCs differentiate to hematopoietic precursors when cocultured with unmodified, nonirradiated donor BM. We did not use any xenogeneic material for this hematopoietic differentiation. Hematopoietic precursors derived from them expressed cell surface antigens CD45/34. When further cultured with hematopoietic growth factors these hematopoietic precursors formed characteristic myeloid, erythroid, and megakaryocyte lineages. Phenotypic CD34+ cells derived from NT-h-ESCs were functionally similar to their counterparts in primary hematopoietic tissues like BM, umbilical cord, and blood. More terminally differentiated hematopoietic cells derived from h-ESCs under these culture conditions also expressed normal surface antigens like glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. We report generation of hematopoietic progenitor cells from h-ESC lines by a SCNT technique, with differentiation into further lineages with structural and functional similarities to their adult counterparts in vivo. This novel alternative source of CD34+ stem cells from h-ESC lines generated without any xenogeneic material might be used to create transplantation tolerance, to implement regenerative medicine, and to treat autoimmune disorders.     

高效绿色荧光蛋白基因修饰前软骨干细胞与骨基质明胶复合体异位成软骨的研究

目的 观察高效绿色荧光蛋白(EGFP)基因修饰前软骨干细胞(PSCs)与骨基质明胶(BMG)复合体的成软骨活性.方法 EGFP基因转染PSCs得到EGFP基因修饰PSCs.将其与BMG的复合体体外培养后植入裸鼠右后肢肌袋中,左后肢植入BMG作对照.定期取材测定移植物的重量、细胞数,观察GFP表达,免疫组织化学检测Ⅱ型胶原表达.结果 EGFP基因修饰PSCs体外培养6周仍有较强GFP表达.移植后1～6 周均有较强GFP表达,3～6周有较强Ⅱ 型胶原表达.移植后2～6周复合体平均重量分别为(0.71±0.07)、(1.35±0.32)、(1.76±0.48)、(1.96±0.54)、(1.89±0.13)g,均比对照组重(P<0.01);1～6周时复合体平均所含细胞总数分别为(40.0±2.1)×106、(67.0±5.6)×106、(139.0±11.2)×106、(169.0±2.9)×106、(173.0±13.5)×106、(169.0±6.9)×106个,均大于对照组(P<0.01).结论 EGFP基因修饰PSCs与BMG复合体能在异位形成软骨组织.

Abstract：

Objective To evaluate the chondrogenesis potential of composite of enhanced green fluorescent protein (EGFP) gene-modified precartilaginous stem cells (PSCs) and bone matrix gelatin (BMG) ectopically. Methods PSCs were transfected with pEGFP by means of lipofectamine. The composite of EGFP gene-modified PSCs and BMG was cultured in vitro, then implanted into the right thigh of athymic mice, and BMG implanted into the left thigh as control group. The harvested implants were weighed, and the number of cells and the expression of GFP were examined. The expression of type Ⅱ collegen was detected by using immunohistochemistry. Results EGFP gene-modified PSCs had high expression of GFP in vitro 6 weeks after purification. The composite had high expression of GFP from 1 to 6 weeks after implantation, and high expression of type Ⅱ collagen from 3 to 6 weeks. The mass of the composite was (0.71±0.07), (1.35±0.32), (1.76±0.48), (1.96±0.54), (1.89±0.13) g from 2 to 6 weeks, respectively, which was significantly greater than that in control group (P<0.01). The total number of cells in the composite was (40.0±2.1)×106, (67.0±5.6)×106, (139.0±11.2)×106, (169.0±2.9)×106, (173.0±13.5)×106, (169.0±6.9)×106 from 1 to 6 weeks, respectively, which was significantly greater than that in control group (P<0.01). Conclusion The composite of EGFP gene-modified PSCs and BMG could generate ectopic cartilaginous tissue.     

绿色荧光高/低转移人肝癌细胞株MHCC97-HG/LG的建立及鉴定

目的建立稳定自发绿色荧光的高、低转移人肝癌细胞株并明确鉴定。方法脂质体转染法将绿色荧光蛋白(GFP)基因分别转染至高、低转移人肝癌细胞株MHCC97-H／L内,经 G418反复筛选并单克隆化,得到两株新细胞株MHCC97-HG／LG,对新细胞株GFP表达稳定性、生物学特性、体内外侵袭转移能力及肿瘤血管生成等进行鉴定。结果 MHCC97-HG／LG在体外培养或裸鼠体内接种36 d后均能稳定表达绿色荧光。MHCC97-HG肝、肺转移率均为100％,较 MHCC97-LG及其父系体内外侵袭转移能力显著增强(P<0．01),其原位肿瘤微血管密度较 MHCC97-LG显著增加[(121．0±15．9)／HP vs(87．0±14．2)／HP,P<0．01]。体视荧光显微镜可实时观察裸鼠体内MHCC97-HG／LG种植瘤生长、转移及血管形成情况。结论 MHCC97-HG／LG及其动物模型在肝癌转移机制、肿瘤血管生成等诸多研究领域可能有较广阔的应用前景。     

绿色荧光蛋白转基因大鼠骨髓间充质干细胞在急性脊髓损伤大鼠中的迁移和分化

目的观察绿色荧光蛋白转基因大鼠来源的骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)在急性脊髓损伤大鼠脊髓组织中的迁移和分化情况。方法全骨髓贴壁培养法培养BMSCs,Allen法制作脊髓损伤大鼠模型,共30只大鼠。于造模1周后进行干预,随机选取造模成功的24只大鼠并随机分为脊髓损伤组、假移植组(生理盐水注射)和细胞移植组(BMSCs注射),每组8只。于移植前、移植后7 d、14 d、21 d、28 d、35 d及42 d进行BBB(Basso,BeattieBresnahan locomotor rating scale)评分。HE染色、免疫荧光观察脊髓损伤的组织修复和BMSCs的迁移分化情况。结果从第14天始,细胞移植组大鼠的BBB评分较脊髓损伤组和假移植组大鼠高,差异具有统计学意义(P0.05)。HE染色显示细胞移植组大鼠的脊髓结构相对完整,液化和囊泡区缩小,炎性细胞减少。免疫荧光显示BMSCs聚集于脊髓损伤处,且能分化为神经元、神经胶质细胞和神经前体细胞。结论 BMSCs能向脊髓损伤部位迁移和聚集,并分化为相应的神经细胞促进脊髓功能恢复。     

Making stem cell lines suitable for transplantation

Human stem cells, progenitor cells, and cell lines have been derived from embryonic, fetal, and adult sources in the search for graft tissue suitable for the treatment of CNS disorders. An increasing number of experimental studies have shown that grafts from several sources survive, differentiate into distinct cell types, and exert positive functional effects in experimental animal models, but little attention has been given to developing cells under conditions of good manufacturing practice (GMP) that can be scaled up for mass treatment. The capacity for continued division of stem cells in culture offers the opportunity to expand their production to meet the widespread clinical demands posed by neurodegenerative diseases. However, maintaining stem cell division in culture long term, while ensuring differentiation after transplantation, requires genetic and/ or oncogenetic manipulations, which may affect the genetic stability and in vivo survival of cells. This review outlines the stages, selection criteria, problems, and ultimately the successes arising in the development of conditionally immortal clinical grade stem cell lines, which divide in vitro, differentiate in vivo, and exert positive functional effects. These processes are specifically exemplified by the murine MHP36 cell line, conditionally immortalized by a temperature-sensitive mutant of the SV40 large T antigen, and cell lines transfected with the c-myc protein fused with a mutated estrogen receptor (c-mycERTAM), regulated by a tamoxifen metabolite, but the issues raised are common to all routes for the development of effective clinical grade cells.     

绿色荧光蛋白转基因小鼠胎肝干细胞在体内外定向分化的潜能

目的探讨绿色荧光蛋白转基因小鼠胎肝干细胞（FLSCs）在体内及体外定向分化的潜能。方法取孕13d绿色荧光蛋白（GFP）转基因C57BL／6J小鼠胚胎肝脏,经机械消化和胶原酶消化获得FLSCs并扩大培养;成熟肝细胞来自于同品系小鼠成体肝脏,通过两步灌注胶原酶消化法提取,流式细胞仪鉴定其干细胞标志物的表达及自我增殖能力,RT—PCR及Westernblot测定AFP表达;分别将FLSCs经肝细胞生长因子（HGF）及TGF-β诱导分化后,Real—timePCR检测Alb、角蛋白CK一7、8、19mRNA以及Westernblot检测Alb、角蛋白CK-7、8、19蛋白在诱导前后的表达变化,鉴定其向肝细胞和胆管细胞双向分化的能力;经尾静脉注射FLSCs到野生型C57BL／6J肝切除小鼠体内,观察其在肝内重构肝组织的能力。计量资料采用t检验,各时相点比较采用重复测量的方差分析,两两比较采用LSD—t检验。结果成功克隆FLSCs细胞。流式细胞仪检测分离的FLSCs表达上皮细胞黏附分子（EpCAM）、CDl33、CD49f干细胞标志物,其阳性表达率分别为78．6％±3．3％、31．7％±2．5％、47．6％±1．8％。AFPmRNA在FLCs和成熟肝细胞中的相对表达量分别为0．640±0．115和0．053±0．012,蛋白相对表达量分别为1．3834±0．265和0．243±0．227,两者比较,差异有统计学意义（t=8．772,5．354,P〈0．05）。超低黏附培养皿培养FLPCs和成熟肝细胞,其形成克隆球数目分别为（234．04±57．0）个和（5．04±2．0）个,两者比较,差异有统计学意义（t=12．711,P〈O．05）。体外HGF诱导FLSCs分化,Alb、CK-8mRNA表达分别在8、10d到达峰值,分别为0．851±0．030和0．771±0．031;两者蛋白水平于第10天达峰值,分别为4．573±0．015和1．562±0．029。体外TGF—B诱导FISCs分化,CK-7、CK-19mRNA在第12天达峰值,分别为15．454±2．152和10．675±1．822;两者蛋白水平于第14天达峰值,分别为3．4234±0．105和1．8924±0．081。在移植有FLSCs的肝切除小鼠体内可检测到表达增强型绿色荧光蛋白（EGFP）的成熟肝细胞及胆管细胞。结论分离的绿色荧光蛋白转基因FLSCs能够稳定表达绿色荧光,具有显著的肝干细胞特征和向肝细胞和胆管细胞双向分化的潜能,且在体内研究中具有良好的示踪性。     

Establishment and characterization of macrophage-like cell lines expressing osteoclast-specific markers

Osteoclasts differentiate from hematopoietic precursors of the monocyte/macrophage lineage to mononuclear preosteoclasts and multinuclear mature osteoclasts. In the present study, we report on the establishment of macrophage-like cell lines from mouse bone marrow by coculturing bone marrow cells with mouse chondrocytes. Isolated clones (MLC-6 and MLC-7 cells) expressed fully differentiated osteoclast markers, such as calcitonin receptors, vitronectin receptors, tartrate-resistant acid phosphatase and vacuolar H⁺-ATPase, in the absence of osteoclast differentiation factor/osteoprotegerin ligand/RANKL/TRANCE, which was essential for osteoclast differentiation. Most clones also maintained expression of a macrophage-associated protein, namely non-specific esterase. Both MLC-6 and MLC-7 cells released cathepsin K into the culture medium. Both clones resolved dentine slices when cocultured with the osteoblast cell line ST2. The cloned cell lines are considered to be useful tools in the study of osteoclast differentiation. Received: October 13, 2000 / Accepted: January 12, 2001     

Stable lines of genetically modified dendritic cells from mouse embryonic stem cells

BACKGROUND: The capacity to activate na?ve T cells sets dendritic cells (DCs) apart from other antigen-presenting cells, making them attractive targets for immune intervention during deleterious immune responses. The inherent resistance of terminally differentiated DCs to conventional strategies for genetic modification has, however, greatly limited our understanding of the molecular mechanisms underlying their function. METHODS AND RESULTS: We report the derivation of long-term cultures of untransformed DCs, uniformly expressing a defined mutant phenotype by the directed differentiation of cloned embryonic stem cells, stably transfected with a reporter gene. Introduction of the gene encoding enhanced green fluorescent protein into pluripotent stem cells demonstrated no observable impact on the phenotype, immunogenicity, or capacity for maturation of DCs differentiated from them. CONCLUSIONS: The production of unlimited numbers of mutant DCs from genetically modified embryonic stem cells paves the way for the systematic elucidation of gene function in this cell type and the rational design of DCs for use in immunotherapy.     

Differentiation of green fluorescent protein-labeled embryonic stem cell-derived neural precursor cells into Thy-1-positive neurons and glia after transplantation into adult rat striatum

OBJECT: The aim of this investigation was to assess new information concerning the capacity of transplanted embryonic stem cell (ESC)-derived neuronal cells to migrate into host brain and to evaluate these cells as a possible source for cell replacement therapy in neurodegenerative disorders such as Parkinson's disease (PD). METHODS: The authors investigated the ability of ESC-derived neural precursor cells to migrate and differentiate in a host striatum by using a D3-derived ESC clone that was transfected stably with a chicken beta-actin cytomegalovirus enhancer-driven green fluorescent protein (GFP)-labeled construct. This procedure allowed easy monitoring of all transplanted cells because of the green fluorescent labeling of donor cells. This approach also afforded easy estimation of cell integration and simultaneous observation of the entire transplanted cell population in relation to immunocytochemically identified neuronal and glial differentiation. After selection of nestin-positive neural precursor cells in a synthetic medium, they were implanted into the striatum of male adult Wistar rats. Their integration was analyzed on morphological studies performed 3 days to 4 weeks posttransplantation. CONCLUSIONS: The investigators found that after transplantation, a subpopulation of GFP-labeled cells differentiated into various neural morphological types that were positive for the mouse-specific Thy-1 antigen, which is known be expressed on neurons, as well as being positive for the astroglial marker glial fibrillary acidic protein. Moreover, GFP-expressing cells that were negative for either of these markers remained close to the injection site, presumably representing other derivatives of the neural lineage. Together, these findings contribute to basic research regarding future transplantation strategies in neurodegenerative diseases such as PD.