小鼠视网膜多潜能干细胞的分离培养和小鼠胚胎干细胞向视网膜色素上皮细胞定向分化的研究
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摘要
研究背景
视网膜色素上皮(Retinal Pigment EPithelium, RPE)具有复杂的结构和特殊的生理机能。RPE能吞噬脱落的光感受器细胞外节膜盘(rodoutersegmeni, ROS),这对光感受器细胞外节的更新及维持正常视觉功能至关重要,其功能障碍会导致严重视网膜疾病和一些严重的遗传性变性眼病,如Best病、Stargardt病、Leber先天性黑曚和年龄相关性黄斑变性(age-related macular degeneration, AMD);等。因此应用RPE细胞移植治疗某些眼底疾病是其中的一个研究热点,目前面临的主要问题是如何获得合适的RPE移植细胞,才能在移植后与宿主残留的视网膜细胞达到最佳的整合效果,以求最大限度地重建患者的视功能。
组织细胞工程尤其是干细胞工程的兴起为视网膜损伤的治疗提供了一种新的途径。极小胚胎样干细胞(very small embryonic-like stem cell, VSEL)是美国路易斯维尔大学Kucia研究小组从小鼠骨髓和人脐带血中分离出scal+lin-CD45-、具有类似胚胎干细胞生物特性的的多能干细胞,Liu等从小鼠视网膜神经上皮中分离了相似的视网膜多潜能干细胞(retinal multipotential stem-like cells, retinalPSC)。已有研究证实：这些多潜能干细胞不仅具有与胚胎干细胞相似的形态学特征和细胞标记物,而且具有胚胎干细胞多分化潜能的特性,在体外可以被诱导分化为内胚层、中胚层、外胚层细胞,潜在分化能力强,若作为组织工程和临床治疗的种子细胞,可避免伦理争议,具有良好的应用前景。通过免疫磁珠分选的方法,我们从新生小鼠小鼠的神经视网膜组织中得到scal+lin-CD45-的同一细胞亚群,通过细胞免疫荧光和流式细胞仪检测其特性,为视网膜移植的供体选择提供新的来源。
当然,目前最广泛的种子细胞来源还是胚胎干细胞(Embryonic stem cell,ES).
胚胎干细胞(Embryonic stem cell,ES)具有无限扩增和多向分化潜能的特点,因此关于其诱导分化的研究成为干细胞研究的热点。如能在体外将胚胎干细胞(ESCs)源源不断地诱导分化成患者所需要的靶细胞(如RPE、光感受器细胞细胞),可望突破视网膜移植中面临的供体来源有限的难题,具有广阔的应用前景。目前,将ESCs体外诱导分化成RPE细胞的研究已经取得了一定的成果,借鉴皮肤黑色素细胞诱导时采用的ST2细胞层和类似的诱导条件,采用PA6间充质细胞系作为饲养细胞层,小鼠ES细胞能分化为呈六角形排列的、胞浆中出现色素颗粒的RPE细胞,但其诱导效率低。除了共培养或组织移植,目前尚无较好获得有效RPE的高效率方法。因此,我们设想建立一种无饲细胞层体外培养的小鼠ES诱导成光感受器和RPE的培养体系,提高ES向RPE诱导效率。
目的
1探讨免疫磁珠分选方法从新生C57小鼠视网膜神经上皮中分离出具有多潜能干细胞特性的scal+lin-CD45-细胞群的可行性。
2研究小鼠视网膜多潜能干细胞与小鼠胚胎干细胞的形态特征及基因表达差异。
3探讨小鼠视网膜多潜能干细胞向RPE分化的途径,建立一种无饲细胞层培养的小鼠ES分步诱导成RPE的高效培养体系。
方法
1小鼠视网膜多潜能干细胞的获取：取初生1天C57小鼠神经视网膜组织制成单细胞悬液,使用免疫磁珠分选的方法先行Lin-阴性分选后行Scal+阳性分选得到scal+lin-CD45-细胞群,使用流式细胞技术和细胞免疫荧光法鉴定其具有极小胚胎样干细胞的特性。
2小鼠视网膜多潜能干细胞和胚胎干细胞的培养：用小鼠成纤维细胞作为饲细胞层,神经干细胞培养基和胚胎干细胞完全培养基分别培养两种细胞,并观察形态特性。
3小鼠胚胎干细胞和视网膜多潜能干细胞及RPE的鉴定：采用碱性磷酸酶染色(alkaline phosphatase, AKP)进行小鼠ES鉴定,采用免疫组织化学方法检测小鼠视网膜多潜能干细胞；用Real Time-PCR方法检测全能性基因Oct4,Nagnog,Sox2在小鼠ES,小鼠视网膜多潜能干细胞与小鼠RPE的表达差异；Reverse Transcription-PCR检测小眼球相关转录因子(microphthalmia-associated transcription factor, MITF)、酪氨酸酶(Tyrosinase, Tyr)、酪氨酸酶相关蛋白1(tyrosinase related protein-1, TRP1),视网膜色素上皮细胞65蛋白(retinal pigment epithelial protein-65, RPE65)在小鼠ES,视网膜多潜能干细胞与RPE的表达差异。
4小鼠ES与诱导产生的RPE基因表达差异性检测：小鼠ES无饲细胞层培养,并添加诱导因子进行26天的诱导方案,采用细胞免疫组织化学方法检测诱导形成的RPE,Real Time-PCR检测ES和RPE的Oct4、Nagnog、Sox2基因表达差异,Reverse Transcription-PCR检测MITF、TYR、TYRP1、RPE65在小鼠ES与RPE的表达差异。
结果
1小鼠视网膜多潜能干细胞分选率：利用免疫磁珠分选的方法从新生小鼠神经视网膜组织中分离出scal+lin-CD45-细胞群,细胞计数分选得率为1.2%。该细胞群干细胞抗原Scal表达率达96.5%,大小为2-6um,形态呈小圆形,核质比高。
2小鼠视网膜多潜能干细胞组织形态学、免疫组化及基因表达：形态呈小圆形,直径2到6um, Scal表达阳性；全能性基因Oct4、Nagnog、Sox2的表达比小鼠ES低,比小鼠视网膜色素上皮细胞高,不表达RPE的标记性基因MITF、TYR、TYRP1、RPE65;小鼠ES呈克隆状生长,排列紧密,细胞集落隆突有立体感,边缘整齐清晰,形似鸟巢。单个细胞胞浆少,细胞核大,核仁明显,界限不清。AKP阳性,全能性基因Oct4、Nagnog、Sox2的高表达。
3小鼠ES向RPE的定向诱导：本实验中尚未从小鼠视网膜多潜能干细胞成功获得RPE；但小鼠ES无饲细胞层培养并添加诱导因子可定向分化为RPE,获得的RPE角蛋白表达阳性,Oct4、Nagnog、Sox2不表达,表达RPE的标记性基因MITF、TYR、TYRP1、RPE65。
结论
1利用免疫磁珠分选的方法可以从新生C57小鼠神经视网膜中成功分离出scal+lin-CD45-细胞群,其分选率为1.2%。
2小鼠视网膜scal+lin-CD45-细胞群具有VSEL的特性。形态呈小圆形,直径2到6um,全能性基因的表达比小鼠ES低,比小鼠视网膜色素细胞高,不表达RPE的标记性基因MITF、TYR、TYRP1、RPE65具有成体干细胞的特性。
3本实验小鼠视网膜多潜能干细胞诱导成RPE存在一定困难,但成功建立了一种小鼠ES无饲细胞层培养并添加诱导因子向RPE诱导的高效培养方案。
BACKGROUND
Retinal Pigment Epithelium is very complex in the construction and its function is special. RPE can phagocytosis the shedding rodoutersegmeni,which is very important in the regeneration of photoreceptor cells and the maintenance of normal vision function. The abnormal Retinal Pigment Epithelium can lead to severe disease of retina and some severe genetic eye diseases, such as Best disease, Stargardt disease and Leber congential blindness, age-related macular degeneration. The RPE cells transplantation in the therapy of eye diseases is a hot area in the future. The problem is that how we can get RPE cells that can be used for transplantation.
The rise of cell sheet engineering especially the stem cell engineering provides a new method for the therapy of retinal damage. The use of stem cell was discussed several times,it is also a hot area in nowadays.
Recently, Kucia group of Louisville university separated a kind of embryonic-like multi-lineage potential cell cells from mouse bone marrow and human umbilical cord blood,named very small embryonic like stem cells. These cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). The morphology of VSEL-SCs is similar to embryonic stem cells and they have the potential of multi-lineage differentiation as the embryonic. VSELSCs can be induced into endoderm, mesoderm and ectoderm cells, they are powerful in the potential of differentiation, and then become a nice candidate for the clinical stem cell. They can also avoid the ethical issues and become useful in the clinical field.
Recently, our group purified a rare population of primitive Scal+/Lin-/CD45-cells from murine bone marrow by employing Magnetic. Based on flow cytometric and gene expression analysis, we want to find a resource of tissue-special stem cells.
Absolutely, the most widely used seed cells are embryonic stem cells.Embryonic stem cells are powerful in the potential of differentiation. Recently, the induction of ESC into RPE in vitro is success in some experiments, The induction of ESC into RPE can be a resource of retina transplantation, which may be widely used in future. But the method of induction is limited.
We use PA6 cells as the feeder cells in the condition similar to the induction of skin melanin cells. The ESC can be differentiate to several kinds of RPE like cells full of pigment when cultured with PA6 cells. There is no better method to obtain RPE except co-culture and tissue transplantation. So we want to build a system that free of feeder cells that can induce ESC to RPE and photoreceptor with high efficiency.
OBJECTIVE
1 To Separate scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic bead sorting.The cells showed the same characterization as the embryonic stem cells.
2 To Analyse the morphology and differences of gene expression of murine retinal multipotential stem-like cells and embryonic stem cells.
3 To explore the induction of RPE from murine retinal multipotential stem-like cells and estabish a stepwise feeder-free culture method of generating RPE from mouse ES cells with high efficiency.
METHODS
1 The purification of mouse retinal retinal multipotential stem-like cells:We separated the tissue of murine nerve-retina from new bron C57 mice and made it to monoplast, then sortted the Lin-cells and then Scal+cells through magnetic beads sorting and got scal+lin-CD45-cells.The FACS and immunofluorescence showed they had the same characterization as the embryonic stem cells.
2 The cultivation of mouse retinal multipotential stem-like cells、mouse ES and RPE:We cultured mouse embryonic stem cells, retinal multipotential stem-like cells with mouse ES medium and NeuroCult Basal Medium, and got the RPE by induction,then observed the characterization.
3 The identification of mouse retinal multipotential stem-like cells,mouse ES and RPE from induction:We identified the ES and mouse retinal multipotential stem-like cells by AKP、immunofluorescence, and we cultured mouse ES with feeder-free method and defined factors for 26 days, then detected embryonic stem cell-derived retinal cells through cell immunohistochemistry, Real Time-PCRand Transcription-PCR.
4 The detection of the differences of gene expression of murine ES、mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:We detected the expression of the three totipotent genes Oct4,Nagnog,Sox2 on the embryonic stem cells, mouse retinal multipotential stem-like cells and retinal pigment epithelium cells through Real Time-PCR. The level of MITF, TYR, RPE65, TYRP1 genes were also analyzed on ES, retinal multipotential stem-like cells and retinal pigment epithelium cells through Reverse Transcription-PCR.
RESULTS
1 The sorting rate of mouse retinal multipotential stem-like cells:We separated scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic beads sorting and got about 1.2% cells.
2 The morphology and immuhistochemisty of mouse retinal multipotential stem-like cells、mouse ES and RPE:Those mouse retinal multipotential stem-like cells expressed Sca1 antigen,their diameter were 3-6um,circle,high nuclear-to-cytoplasm ratio.The mouse ES showed typical morphology and expressed AKP positive. The RPE expressed specific markers.
3 The differences of gene expression of murine ES、mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:Real Time-PCR showed that the three totipotent genes Oct4,Nagnog,Sox2 expressed on embryonic stem cells, retinal multipotential stem-like cells but not on retinal pigment epithelium cells.Reverse-Transcription PCR showed that MITF, TYR, TYRP1 RPE65genes had a different expression level on them.
4 The induction of RPE from mouse ES:We got RPE cells from mouse ES but not from mouse retinal multipotential stem-like cells through our protocal. The embryonic stem cell-derived retinal cells expressed specific markers.
CONCLUSION
1 We separated retinal multipotential stem-like cells from the tissue of neonatal murine retina successfully.The cells showed the same characterization as the embryonic stem cells.
2 The expression level of potential genes on retinal multipotential stem-like cells was lower than embryonic cells but much higher than RPE,showed the characterization of adult stem cell.
3 We failed to induce mouse retinal multipotential stem-like cells into RPE,but have established a stepwise feeder-free culture method of generating RPE from mouse ES cells in vitro with high efficiency.
视网膜色素上皮(Retinal Pigment EPithelium, RPE)具有复杂的结构和特殊的生理机能。RPE能吞噬脱落的光感受器细胞外节膜盘(rodoutersegmeni, ROS),这对光感受器细胞外节的更新及维持正常视觉功能至关重要,其功能障碍会导致严重视网膜疾病和一些严重的遗传性变性眼病,如Best病、Stargardt病、Leber先天性黑曚和年龄相关性黄斑变性(age-related macular degeneration, AMD);等。因此应用RPE细胞移植治疗某些眼底疾病是其中的一个研究热点,目前面临的主要问题是如何获得合适的RPE移植细胞,才能在移植后与宿主残留的视网膜细胞达到最佳的整合效果,以求最大限度地重建患者的视功能。
组织细胞工程尤其是干细胞工程的兴起为视网膜损伤的治疗提供了一种新的途径。极小胚胎样干细胞(very small embryonic-like stem cell, VSEL)是美国路易斯维尔大学Kucia研究小组从小鼠骨髓和人脐带血中分离出scal+lin-CD45-、具有类似胚胎干细胞生物特性的的多能干细胞,Liu等从小鼠视网膜神经上皮中分离了相似的视网膜多潜能干细胞(retinal multipotential stem-like cells, retinalPSC)。已有研究证实：这些多潜能干细胞不仅具有与胚胎干细胞相似的形态学特征和细胞标记物,而且具有胚胎干细胞多分化潜能的特性,在体外可以被诱导分化为内胚层、中胚层、外胚层细胞,潜在分化能力强,若作为组织工程和临床治疗的种子细胞,可避免伦理争议,具有良好的应用前景。通过免疫磁珠分选的方法,我们从新生小鼠小鼠的神经视网膜组织中得到scal+lin-CD45-的同一细胞亚群,通过细胞免疫荧光和流式细胞仪检测其特性,为视网膜移植的供体选择提供新的来源。
当然,目前最广泛的种子细胞来源还是胚胎干细胞(Embryonic stem cell,ES).
胚胎干细胞(Embryonic stem cell,ES)具有无限扩增和多向分化潜能的特点,因此关于其诱导分化的研究成为干细胞研究的热点。如能在体外将胚胎干细胞(ESCs)源源不断地诱导分化成患者所需要的靶细胞(如RPE、光感受器细胞细胞),可望突破视网膜移植中面临的供体来源有限的难题,具有广阔的应用前景。目前,将ESCs体外诱导分化成RPE细胞的研究已经取得了一定的成果,借鉴皮肤黑色素细胞诱导时采用的ST2细胞层和类似的诱导条件,采用PA6间充质细胞系作为饲养细胞层,小鼠ES细胞能分化为呈六角形排列的、胞浆中出现色素颗粒的RPE细胞,但其诱导效率低。除了共培养或组织移植,目前尚无较好获得有效RPE的高效率方法。因此,我们设想建立一种无饲细胞层体外培养的小鼠ES诱导成光感受器和RPE的培养体系,提高ES向RPE诱导效率。
目的
1探讨免疫磁珠分选方法从新生C57小鼠视网膜神经上皮中分离出具有多潜能干细胞特性的scal+lin-CD45-细胞群的可行性。
2研究小鼠视网膜多潜能干细胞与小鼠胚胎干细胞的形态特征及基因表达差异。
3探讨小鼠视网膜多潜能干细胞向RPE分化的途径,建立一种无饲细胞层培养的小鼠ES分步诱导成RPE的高效培养体系。
方法
1小鼠视网膜多潜能干细胞的获取：取初生1天C57小鼠神经视网膜组织制成单细胞悬液,使用免疫磁珠分选的方法先行Lin-阴性分选后行Scal+阳性分选得到scal+lin-CD45-细胞群,使用流式细胞技术和细胞免疫荧光法鉴定其具有极小胚胎样干细胞的特性。
2小鼠视网膜多潜能干细胞和胚胎干细胞的培养：用小鼠成纤维细胞作为饲细胞层,神经干细胞培养基和胚胎干细胞完全培养基分别培养两种细胞,并观察形态特性。
3小鼠胚胎干细胞和视网膜多潜能干细胞及RPE的鉴定：采用碱性磷酸酶染色(alkaline phosphatase, AKP)进行小鼠ES鉴定,采用免疫组织化学方法检测小鼠视网膜多潜能干细胞；用Real Time-PCR方法检测全能性基因Oct4,Nagnog,Sox2在小鼠ES,小鼠视网膜多潜能干细胞与小鼠RPE的表达差异；Reverse Transcription-PCR检测小眼球相关转录因子(microphthalmia-associated transcription factor, MITF)、酪氨酸酶(Tyrosinase, Tyr)、酪氨酸酶相关蛋白1(tyrosinase related protein-1, TRP1),视网膜色素上皮细胞65蛋白(retinal pigment epithelial protein-65, RPE65)在小鼠ES,视网膜多潜能干细胞与RPE的表达差异。
4小鼠ES与诱导产生的RPE基因表达差异性检测：小鼠ES无饲细胞层培养,并添加诱导因子进行26天的诱导方案,采用细胞免疫组织化学方法检测诱导形成的RPE,Real Time-PCR检测ES和RPE的Oct4、Nagnog、Sox2基因表达差异,Reverse Transcription-PCR检测MITF、TYR、TYRP1、RPE65在小鼠ES与RPE的表达差异。
结果
1小鼠视网膜多潜能干细胞分选率：利用免疫磁珠分选的方法从新生小鼠神经视网膜组织中分离出scal+lin-CD45-细胞群,细胞计数分选得率为1.2%。该细胞群干细胞抗原Scal表达率达96.5%,大小为2-6um,形态呈小圆形,核质比高。
2小鼠视网膜多潜能干细胞组织形态学、免疫组化及基因表达：形态呈小圆形,直径2到6um, Scal表达阳性；全能性基因Oct4、Nagnog、Sox2的表达比小鼠ES低,比小鼠视网膜色素上皮细胞高,不表达RPE的标记性基因MITF、TYR、TYRP1、RPE65;小鼠ES呈克隆状生长,排列紧密,细胞集落隆突有立体感,边缘整齐清晰,形似鸟巢。单个细胞胞浆少,细胞核大,核仁明显,界限不清。AKP阳性,全能性基因Oct4、Nagnog、Sox2的高表达。
3小鼠ES向RPE的定向诱导：本实验中尚未从小鼠视网膜多潜能干细胞成功获得RPE；但小鼠ES无饲细胞层培养并添加诱导因子可定向分化为RPE,获得的RPE角蛋白表达阳性,Oct4、Nagnog、Sox2不表达,表达RPE的标记性基因MITF、TYR、TYRP1、RPE65。
结论
1利用免疫磁珠分选的方法可以从新生C57小鼠神经视网膜中成功分离出scal+lin-CD45-细胞群,其分选率为1.2%。
2小鼠视网膜scal+lin-CD45-细胞群具有VSEL的特性。形态呈小圆形,直径2到6um,全能性基因的表达比小鼠ES低,比小鼠视网膜色素细胞高,不表达RPE的标记性基因MITF、TYR、TYRP1、RPE65具有成体干细胞的特性。
3本实验小鼠视网膜多潜能干细胞诱导成RPE存在一定困难,但成功建立了一种小鼠ES无饲细胞层培养并添加诱导因子向RPE诱导的高效培养方案。
BACKGROUND
Retinal Pigment Epithelium is very complex in the construction and its function is special. RPE can phagocytosis the shedding rodoutersegmeni,which is very important in the regeneration of photoreceptor cells and the maintenance of normal vision function. The abnormal Retinal Pigment Epithelium can lead to severe disease of retina and some severe genetic eye diseases, such as Best disease, Stargardt disease and Leber congential blindness, age-related macular degeneration. The RPE cells transplantation in the therapy of eye diseases is a hot area in the future. The problem is that how we can get RPE cells that can be used for transplantation.
The rise of cell sheet engineering especially the stem cell engineering provides a new method for the therapy of retinal damage. The use of stem cell was discussed several times,it is also a hot area in nowadays.
Recently, Kucia group of Louisville university separated a kind of embryonic-like multi-lineage potential cell cells from mouse bone marrow and human umbilical cord blood,named very small embryonic like stem cells. These cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). The morphology of VSEL-SCs is similar to embryonic stem cells and they have the potential of multi-lineage differentiation as the embryonic. VSELSCs can be induced into endoderm, mesoderm and ectoderm cells, they are powerful in the potential of differentiation, and then become a nice candidate for the clinical stem cell. They can also avoid the ethical issues and become useful in the clinical field.
Recently, our group purified a rare population of primitive Scal+/Lin-/CD45-cells from murine bone marrow by employing Magnetic. Based on flow cytometric and gene expression analysis, we want to find a resource of tissue-special stem cells.
Absolutely, the most widely used seed cells are embryonic stem cells.Embryonic stem cells are powerful in the potential of differentiation. Recently, the induction of ESC into RPE in vitro is success in some experiments, The induction of ESC into RPE can be a resource of retina transplantation, which may be widely used in future. But the method of induction is limited.
We use PA6 cells as the feeder cells in the condition similar to the induction of skin melanin cells. The ESC can be differentiate to several kinds of RPE like cells full of pigment when cultured with PA6 cells. There is no better method to obtain RPE except co-culture and tissue transplantation. So we want to build a system that free of feeder cells that can induce ESC to RPE and photoreceptor with high efficiency.
OBJECTIVE
1 To Separate scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic bead sorting.The cells showed the same characterization as the embryonic stem cells.
2 To Analyse the morphology and differences of gene expression of murine retinal multipotential stem-like cells and embryonic stem cells.
3 To explore the induction of RPE from murine retinal multipotential stem-like cells and estabish a stepwise feeder-free culture method of generating RPE from mouse ES cells with high efficiency.
METHODS
1 The purification of mouse retinal retinal multipotential stem-like cells:We separated the tissue of murine nerve-retina from new bron C57 mice and made it to monoplast, then sortted the Lin-cells and then Scal+cells through magnetic beads sorting and got scal+lin-CD45-cells.The FACS and immunofluorescence showed they had the same characterization as the embryonic stem cells.
2 The cultivation of mouse retinal multipotential stem-like cells、mouse ES and RPE:We cultured mouse embryonic stem cells, retinal multipotential stem-like cells with mouse ES medium and NeuroCult Basal Medium, and got the RPE by induction,then observed the characterization.
3 The identification of mouse retinal multipotential stem-like cells,mouse ES and RPE from induction:We identified the ES and mouse retinal multipotential stem-like cells by AKP、immunofluorescence, and we cultured mouse ES with feeder-free method and defined factors for 26 days, then detected embryonic stem cell-derived retinal cells through cell immunohistochemistry, Real Time-PCRand Transcription-PCR.
4 The detection of the differences of gene expression of murine ES、mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:We detected the expression of the three totipotent genes Oct4,Nagnog,Sox2 on the embryonic stem cells, mouse retinal multipotential stem-like cells and retinal pigment epithelium cells through Real Time-PCR. The level of MITF, TYR, RPE65, TYRP1 genes were also analyzed on ES, retinal multipotential stem-like cells and retinal pigment epithelium cells through Reverse Transcription-PCR.
RESULTS
1 The sorting rate of mouse retinal multipotential stem-like cells:We separated scal+lin-CD45-cells from the tissue of neonatal murine retina through the method of magnetic beads sorting and got about 1.2% cells.
2 The morphology and immuhistochemisty of mouse retinal multipotential stem-like cells、mouse ES and RPE:Those mouse retinal multipotential stem-like cells expressed Sca1 antigen,their diameter were 3-6um,circle,high nuclear-to-cytoplasm ratio.The mouse ES showed typical morphology and expressed AKP positive. The RPE expressed specific markers.
3 The differences of gene expression of murine ES、mouse retinal multipotential stem-like cells and RPE and the murine ES and ES-induced RPE:Real Time-PCR showed that the three totipotent genes Oct4,Nagnog,Sox2 expressed on embryonic stem cells, retinal multipotential stem-like cells but not on retinal pigment epithelium cells.Reverse-Transcription PCR showed that MITF, TYR, TYRP1 RPE65genes had a different expression level on them.
4 The induction of RPE from mouse ES:We got RPE cells from mouse ES but not from mouse retinal multipotential stem-like cells through our protocal. The embryonic stem cell-derived retinal cells expressed specific markers.
CONCLUSION
1 We separated retinal multipotential stem-like cells from the tissue of neonatal murine retina successfully.The cells showed the same characterization as the embryonic stem cells.
2 The expression level of potential genes on retinal multipotential stem-like cells was lower than embryonic cells but much higher than RPE,showed the characterization of adult stem cell.
3 We failed to induce mouse retinal multipotential stem-like cells into RPE,but have established a stepwise feeder-free culture method of generating RPE from mouse ES cells in vitro with high efficiency.
引文
[1]Eekardt C, EekardtU, Corirad HG; Macular rotation with and without counter-rotation of the globe in Patients Withage-related macular degeneration. Graefes Arch Clin EXP OPhthalmol2001;237-313
[2]Petrukhin K, Koisti, MJ, BakallB, etal. Identification of the gene responsible for BestMacular dystrophy. NatGenet,2008;19:241-247
[3]Marnnorstein, AD, Marmorstein, LY, Rayborn etal. Bestrophin the product of the Best Vitelliform macular dystrophy gene(VMDZ), localizes to the basolateral plasma membrane of the retinal Pigment epithelium. Proc Nat Acad Sei USA 2000:97:12758-12763
[4]Yong qing Liu1.2.6, Ling Gao1,4,6, Ewa K. et al. Identification of Small Sca-1+, Lin-, CD45- Multipotential Cells in the Neonatal Murine Retina.Stem Cell Reviews and Reports.2007,6:189-192
[5]Fumitaka Osakadal, Hanako Ikedal, Yoshiki Sasai, Masayo Takahashi Stepwise differentiation of pluripotent stem cells intoretinal cells. Nature protocol 2009:10.1038.
[6]Chaeko DM, DasA ZhaoX, etal. Trans Plantation of ocular stem cells:the role of injury in incorporation and differentiation of grafted cells in the retina.Vision Res,20038,43(8):937-946.
[7]W. Cao 1*, J. Tombran-Tink 2, W. Chen 1, D. Mrazek 2, R. Elias 1, J.F. McGinnis Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death Journal of Neuroscience Research,1999,57:789-800:
[8]JeffreyR.Harris,GaryA,Brown,etal.BoneMarrow-Derived Cells Home to and Regener Retinal Piglnent Epithelium after Injury.Invest OPhthalmol VisSci.2006;47:2108-113
[9]PittengerMF, MackayAM, BeekSC, etal.MultolineagePotentialofadulthuman mesenchymalstemeells[J].Seienee,1999:284(2):143
[10]Kucia M, Wysoczynski M, Ratajczak J, et al. Identification of very small embryonic like (VSEL) stem cells in bone marrow [J]. Cell Tissue Res, 2008,331 (1):1252134.
[11]Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4(+) SSEA-4(+) Oct-4(+) very small embryonic-like cells purified from human cord blood-preliminary report. Leukemia 2007;21:297-303.
[12]Mariusz Z. Ratajczak,, Ewa K. Zuba-Surmal, and Magda Kucial Very Small Embryonic-Like (VSEL) Stem Cells:Purification from Adult Organs, Characterization, and Biological Significance. Stem Cell Reviews and Reports.2008,6:89-99
[13]Y Jiang B.Vaessen,T.Lenvik,etal. Multi Potent Progenitor cells can be isolated from postnatal murine bone marrow muscle and brain Exp.Hematol.2002:30:896-904
[14]A Hermann,R.Gastl,S.Liebau,M.O.PoPa,E.Cient generation of neural stem cell-like cellsFrom adult human bone marrow stromal cells [J]. CellSei.2004:117:4411-422
[15]Y Jiang B.N.Jahagirdar R.L.Reinhardt, etal. Pluri Potency of mesenehymal stem cells derived from adult marrow,Nature.2002:418:41-9
[16]D Woodbury, E.J.Sehwarz, D.J.ProckoP, etal.Adultratandhuman bonemarrowstromal eellsdiereniiateinioneurons, J.Neurosci.Res.2000;61:364-70
[17]Albert O. Edwards, Robert Ritter,Ⅲ,1 Kenneth J. Abel, Alisa Manning,Carolien Panhuysen, Lindsay A. Farrer Complement Factor H Polymorphism and Age-Related Macular Degeneration,Science:2005,5:421-424
[18]Howell JC, Lee WH, Morrison P, Zhong J, Yoder MC, Srour EF. Pluripotent stem cells identified in multiple murine tissues. Ann N Y Acad Sci.2003; 996:158-73.
[19]Justine R. Smith 1*, Ralph D. Levinson Differential efficacy of tumor necrosis factor inhibition in the management of inflammatory eye disease and associated rheumatic diseaseArthritis Care & Research,2001:252-257
[20]Pittenger MF, MackayAM, BeekSC, etal.Multolineage Potential of adult human Mesenchymal stem cells[J].Seienee,1999:284(2):143
[21]Dennis JE,CharbordP.Origin and ifferentiation of human and murine stroma.StemCells.2002:20(3):205-14
[22]AnitaM, RallieriC, RodolfoQ.Clonal mesene hymal progenitors from human bone marrowdifferentiate in vitro according to a hierarehieal model. JournalofCellScience.2000:113:1161-1166
[23]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71-74.
[24]Shi Q, Rafi S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362-367.
[25]Gronthos S, Liu ZJ, Xiao M, et al. Molecular and cellular characterization of highly purified stromal stem cells derived form human bone marrow. J Cell Sci 2003;116:1827-1835.
[26]Ratajczak MZ, Kucia M, Ratajczak J et al. A multi-instrumental approach to identify and purify very small embryonic like stem cells (VSELs) from adult tissues. Micron 2009:40:386-393.
[27]Zuba-Surma EK, Kucia M, Abdel-Latif A, et al. Morphological characterization of very small embryonic-like stem cells (VSELs) by image stream system analysis. J Cell Mol Med 2008;12:292-303.
[28]Kucia M, Reca R, Campbell FR, Zuba-Surma E,Majka M, Ratajczak J, Ratajczak MZ. A populationof very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+stem cells identified in adult bone marrow.Leukemia.2006; 20:857-69.
[29]Bharti K, Nguyen M.-TT, Skuntz S, et al. The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye. Pig, Cell Res 2006;19:380-394.
[30]冯钰.色素形成相关蛋白在胚胎大鼠视网膜发育中的表达：[硕士学位论文].长沙：中南大学,2009
[31]Hess DC, Abe T, Hill WD, et al. Hematopoietic origin of microglial and perivascular cells in brain.Exp Neurol 2004; 186:134-144. [PubMed:15026252]
[32]Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain:cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779-1782. [PubMed:11099419]
[33]Hasegawa T, Kosaki A, Shimizu K, et al. Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats. Exp Neurol2006; 199:274-280. [PubMed: 16337192]
[34]Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243-252. [PubMed: 14722616]
[35]Song YS, Ryu YH, Choi SR, Kim JC. The involvement of adult stem cells originated from bone marrow in the pathogenesis of pterygia. Yonsei Med J 2005;46:687-692. [PubMed:16259068]
[36]Virchow R. Editorial Archive fuer pathologische Anatomie und Physiologie fuer klinische Medizin1855;8:23-54.
[37]ughton J, Stoicov C, Nomura S, et al. Gastric cancer originating from bone marrow-derived cells.Science 2004:306.
[38]Kucia M, Ratajczak J, Ratajczak MZ. Bone marrow as a source of circulating CXCR4+tissuecommitted stem cells. Biol Cell 2005;97:133-146. [PubMed: 15656779]
[39]Wysoczynski M, Kucia M, Zuba-Surma EK, Wu W, Ratajczak MZ, Ratajczak J. An in vivo evidence that the CD45negative adult marrow-derived CXCR4+ SSEA-1+Oct-4+Very Small Embryonic Like (VSEL) stem cells may differentiate into CD45positive long term repopulating hematopoietic stem cells. ASH Meeting:Blood 2007:505.
[40]Jones RJ, Collector MI, Barber JP, et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity. Blood 1996;88:487-91. [PubMed:8695796]
[41]Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990;347:188-189. [PubMed:2395470]
[42]Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329-1337. [PubMed:8946831]
[43]Dawn B, TiwariS, Kucia M J, et al. Transplantation of bone Marrow derived very small embryonic2like stem cell s attenuates left vent ricular dysfunction and remodeling after myocardial in farction [J]. Stem Cells,2008,26 (6):164621655.
[44]Zuba2Surma E K, Kucia M, Dawn B, et al. Bone marrow2derived pluripotent very small embryonic2like stem cells (VSELs) are mobilized after acute myocardial infarction [J]. J Mol Cell Cardiol,2008,44 (5):8652873.
[45]Kucia M J, Wysoczynski M, Wu W, et al. Evidence t hat very small embryonic like stem cells are mobilized into peripheral blood [J]. Stem Cells,2008,26 (8):208322092.
[46]Ratajczak M Z, Zuba2Surma E K, Shin D M, et al. Very small embryonic2like (VSEL) stem cells in adult organs and t heir potential role in rejuvenation of tissues and longevity [J]. Exp Gerontol,2008,43 (11):100921017.
[1]Colter D C, Sekiya I, Prockop D J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cell s in colonies of human marrow stromal cell s [J]. Proc Natl Acad SciUSA,2001,98 (14):7841-7845.
[2]Jiang Y, J ahagirdar B N, Reinhardt R L, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature,2002,418 (6893):41-49.
[3]DpIppolito G, Diabira S, Howard G A, et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential [J]. J Cell Sci 2004,117(14):2971-2981.
[4]Kogler G, Sensken S, Airey J A, et al. A new human somatic stem cell from placental cord blood wit h int rinsic pluripotent differentiation potential [J]. J Exp Med,2004,200 (2):123-135.
[5]Zuba-Surma E K, Kucia M, Ratajczak J, et al."Small stem cells" in adult tissues:Very small embryonic2like stem cells stand up[J]. Cytomet ry A 2008,75A(1):4-13.
[6]Ratajczak M Z, Kucia M, Reca R, et al. Stem cell plasticity re visited: CXCR4-positive cell s expressing mRNA for early muscle, liver and neural cells'hide out'in t he bone marrow [J]. Leukemia,2004,8 (1):29-40.
[7]Kucia M, Ratajczak J, Ratajczak M Z. Are bone marrow stem-cells plastic or heterogeneous that is t he question [J]. Exp Hematol,2005,33 (6):613-623.
[8]Kucia M, Wysoczynski M, Ratajczak J, et al. Identification of very small embryonic like (VSEL) stem cells in bone marrow [J].Cell Tissue Res 2008,331 (1):125-134.
[9]Kucia M, Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4+SSEA-4+Oct-4+very small embryonic like cells purified from human cord blood preliminary report [J] Leukemia,2007,21 (2):297-303.
[10]Halasa M, Baskiewicz Masiuk M, Dabkowska E, et al. An efficient two step method to purify very small embryonic like(VSEL) stem cells from umbilical cord blood (UCB) [J]. Folia Histochem Cytobiol,2008,46 (2):239-243.
[11]Kucia M, Zuba-Surma E K, Wysoczynski M, et al. Adult marrow derived very small embryonic-like stem cells and tissue engineering [J]. Expert Opin Biol Ther,2007,7 (10):1499-1514.
[12]Zuba Surma E K, Kucia M, Wu W, et al. Very small embryonic like stem cells are present in adult murine organs:Image Stream based morphological analysis and distribution studies [J]. Cytom2 et ry A,2008,73A(12):1116-1127.
[13]Ratajczak M Z, Machalinski B, Wojakowski W,et al. A hypothesis for an embryonic origin of pluripotent Oct-4 (+) stem cells in adult bone marrow and other tissues [J]. Leukemia,2007,21(5):860-867.
[14]Kucia M, Wu W, Ratajczak M Z. Bone marrow-derived very small embryonic like stem cells:Their developmental origin and biological significance [J]. DevDyn,2007,236 (12):3309-3320.
[15]Zuba Surma E K, Kucia M, Abdel-Latif A, et al. Morphological characterization of very small embryonic21ike stem cell s (VSELs) by Image Stream system analysis [J]. J Cell Mol Med,2008,12 (1):292-303.
[16]Kucia M, Reca R, Campbell F R, et al. A population of very small embryonic like (VSEL) CXCR4 (+) SSEA-1 (+) Oct-4+stem cells identified in adult bone marrow [J]. Leukemia,2006,20 (5):857-869.
[17]Ratajczak J, Kucia M, Zuba2Surma E, et al. The CD45-Linadult Marrow derived CXCR4+SSEA-1+Oct-4+very small embryonic like (VSEL-SC) stem cells form in vitro spheres which may differentiate into CD45+hematopoietic stem cell s [R]. ASH,48 the annual meeting. Orlando, USA(10):Abstract 280.
[18]Dawn B, TiwariS, Kucia M J, et al. Transplantation of bone Marrow derived very small embryonic-like stem cell s attenuates left vent ricular dysfunction and remodeling after myocardial in farction [J]. Stem Cells,2008,26 (6):1646-1655.
[19]Zuba Surma E K, Kucia M, Dawn B, et al. Bone marrow-derived pluripotent very small embryonic like stem cells (VSELs) are mobilized after acute myocardial infarction [J]. J Mol Cell Cardiol,2008,44 (5):865-873.
[20]Kucia M J, Wysoczynski M, Wu W, et al. Evidence t hat very small embryonic like stem cells are mobilized into peripheral blood [J]. Stem Cells,2008,26 (8):2083-2092.
[21]Ratajczak M Z, Zuba-Surma E K, Shin D M, et al. Very small Embryonic like (VSEL) stem cells in adult organs and t heir potential role in rejuvenation of tissues and longevity [J]. Exp Gerontol,2008,43 (11):1009-1017.
[22]Lamoury F M, Croitoru-Lamoury J, Brew B J. Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct24 and Rex21 [J]. Cytot herapy,2006,8(3):228-242.
[23]Pochampally R R, Smith J R, Ylostalo J, et al. Serum deprivation of human marrow stromal cells (hMSCs) select s for a subpopulation of early progenitor cells with enhanced expression of OCT24 and other embryonic genes [J]. Blood, 2004,103(5):1647-1652.
[24]Zeng L, Rahrmann E, Hu Q, et al. Multipotent adult progenitor cells f rom swine bone marrow [J]. Stem Cells,2006,24 (11):2355-2366.
[25]Pallante B A, Duignan I, Okin D, et al. Bone marrow Oct3/4+cells differentiate into cardiac myocytes via age2dependent paracrine mechanisms [J]. Circ Res,2007,100(1):1-11.
[26]Anjos-Afonso F, Bonnet D. Nonhematopoietic/endot helial SSEA21+cells define t he most primitive progenitors in t he adult murine bone marrow mesenchymal compartment [J]. Blood,2007,109(3):1298-1306.
[27]Ratajczak M Z, Zuba-Surma E K, Machalinski B, et al. Very small embryonic like (VSEL) stem cells:purification f rom adult organs, characterization, and biological significance [J]. StemCell Rev,2008,4 (2):89-99.
[28]Zuba-Surma E K, Wu W, Ratajczak J, et al. Very small embryonic like stem cells in adult tissues2Potential implications for aging[J]. Mech Ageing Dev, 2009,130 (122):58-66.
[29]Kucia M, Zuba-Surma E, Wysoczynski M, et al. Physiological and pathological consequences of identification of very small embryonic like (VSEL) stem cells in adult bone marrow [J]. J Physiol Pharmacol,2006,57 (5):5-18.
[2]Petrukhin K, Koisti, MJ, BakallB, etal. Identification of the gene responsible for BestMacular dystrophy. NatGenet,2008;19:241-247
[3]Marnnorstein, AD, Marmorstein, LY, Rayborn etal. Bestrophin the product of the Best Vitelliform macular dystrophy gene(VMDZ), localizes to the basolateral plasma membrane of the retinal Pigment epithelium. Proc Nat Acad Sei USA 2000:97:12758-12763
[4]Yong qing Liu1.2.6, Ling Gao1,4,6, Ewa K. et al. Identification of Small Sca-1+, Lin-, CD45- Multipotential Cells in the Neonatal Murine Retina.Stem Cell Reviews and Reports.2007,6:189-192
[5]Fumitaka Osakadal, Hanako Ikedal, Yoshiki Sasai, Masayo Takahashi Stepwise differentiation of pluripotent stem cells intoretinal cells. Nature protocol 2009:10.1038.
[6]Chaeko DM, DasA ZhaoX, etal. Trans Plantation of ocular stem cells:the role of injury in incorporation and differentiation of grafted cells in the retina.Vision Res,20038,43(8):937-946.
[7]W. Cao 1*, J. Tombran-Tink 2, W. Chen 1, D. Mrazek 2, R. Elias 1, J.F. McGinnis Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death Journal of Neuroscience Research,1999,57:789-800:
[8]JeffreyR.Harris,GaryA,Brown,etal.BoneMarrow-Derived Cells Home to and Regener Retinal Piglnent Epithelium after Injury.Invest OPhthalmol VisSci.2006;47:2108-113
[9]PittengerMF, MackayAM, BeekSC, etal.MultolineagePotentialofadulthuman mesenchymalstemeells[J].Seienee,1999:284(2):143
[10]Kucia M, Wysoczynski M, Ratajczak J, et al. Identification of very small embryonic like (VSEL) stem cells in bone marrow [J]. Cell Tissue Res, 2008,331 (1):1252134.
[11]Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4(+) SSEA-4(+) Oct-4(+) very small embryonic-like cells purified from human cord blood-preliminary report. Leukemia 2007;21:297-303.
[12]Mariusz Z. Ratajczak,, Ewa K. Zuba-Surmal, and Magda Kucial Very Small Embryonic-Like (VSEL) Stem Cells:Purification from Adult Organs, Characterization, and Biological Significance. Stem Cell Reviews and Reports.2008,6:89-99
[13]Y Jiang B.Vaessen,T.Lenvik,etal. Multi Potent Progenitor cells can be isolated from postnatal murine bone marrow muscle and brain Exp.Hematol.2002:30:896-904
[14]A Hermann,R.Gastl,S.Liebau,M.O.PoPa,E.Cient generation of neural stem cell-like cellsFrom adult human bone marrow stromal cells [J]. CellSei.2004:117:4411-422
[15]Y Jiang B.N.Jahagirdar R.L.Reinhardt, etal. Pluri Potency of mesenehymal stem cells derived from adult marrow,Nature.2002:418:41-9
[16]D Woodbury, E.J.Sehwarz, D.J.ProckoP, etal.Adultratandhuman bonemarrowstromal eellsdiereniiateinioneurons, J.Neurosci.Res.2000;61:364-70
[17]Albert O. Edwards, Robert Ritter,Ⅲ,1 Kenneth J. Abel, Alisa Manning,Carolien Panhuysen, Lindsay A. Farrer Complement Factor H Polymorphism and Age-Related Macular Degeneration,Science:2005,5:421-424
[18]Howell JC, Lee WH, Morrison P, Zhong J, Yoder MC, Srour EF. Pluripotent stem cells identified in multiple murine tissues. Ann N Y Acad Sci.2003; 996:158-73.
[19]Justine R. Smith 1*, Ralph D. Levinson Differential efficacy of tumor necrosis factor inhibition in the management of inflammatory eye disease and associated rheumatic diseaseArthritis Care & Research,2001:252-257
[20]Pittenger MF, MackayAM, BeekSC, etal.Multolineage Potential of adult human Mesenchymal stem cells[J].Seienee,1999:284(2):143
[21]Dennis JE,CharbordP.Origin and ifferentiation of human and murine stroma.StemCells.2002:20(3):205-14
[22]AnitaM, RallieriC, RodolfoQ.Clonal mesene hymal progenitors from human bone marrowdifferentiate in vitro according to a hierarehieal model. JournalofCellScience.2000:113:1161-1166
[23]Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71-74.
[24]Shi Q, Rafi S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362-367.
[25]Gronthos S, Liu ZJ, Xiao M, et al. Molecular and cellular characterization of highly purified stromal stem cells derived form human bone marrow. J Cell Sci 2003;116:1827-1835.
[26]Ratajczak MZ, Kucia M, Ratajczak J et al. A multi-instrumental approach to identify and purify very small embryonic like stem cells (VSELs) from adult tissues. Micron 2009:40:386-393.
[27]Zuba-Surma EK, Kucia M, Abdel-Latif A, et al. Morphological characterization of very small embryonic-like stem cells (VSELs) by image stream system analysis. J Cell Mol Med 2008;12:292-303.
[28]Kucia M, Reca R, Campbell FR, Zuba-Surma E,Majka M, Ratajczak J, Ratajczak MZ. A populationof very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+stem cells identified in adult bone marrow.Leukemia.2006; 20:857-69.
[29]Bharti K, Nguyen M.-TT, Skuntz S, et al. The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye. Pig, Cell Res 2006;19:380-394.
[30]冯钰.色素形成相关蛋白在胚胎大鼠视网膜发育中的表达：[硕士学位论文].长沙：中南大学,2009
[31]Hess DC, Abe T, Hill WD, et al. Hematopoietic origin of microglial and perivascular cells in brain.Exp Neurol 2004; 186:134-144. [PubMed:15026252]
[32]Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR. Turning blood into brain:cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779-1782. [PubMed:11099419]
[33]Hasegawa T, Kosaki A, Shimizu K, et al. Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats. Exp Neurol2006; 199:274-280. [PubMed: 16337192]
[34]Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243-252. [PubMed: 14722616]
[35]Song YS, Ryu YH, Choi SR, Kim JC. The involvement of adult stem cells originated from bone marrow in the pathogenesis of pterygia. Yonsei Med J 2005;46:687-692. [PubMed:16259068]
[36]Virchow R. Editorial Archive fuer pathologische Anatomie und Physiologie fuer klinische Medizin1855;8:23-54.
[37]ughton J, Stoicov C, Nomura S, et al. Gastric cancer originating from bone marrow-derived cells.Science 2004:306.
[38]Kucia M, Ratajczak J, Ratajczak MZ. Bone marrow as a source of circulating CXCR4+tissuecommitted stem cells. Biol Cell 2005;97:133-146. [PubMed: 15656779]
[39]Wysoczynski M, Kucia M, Zuba-Surma EK, Wu W, Ratajczak MZ, Ratajczak J. An in vivo evidence that the CD45negative adult marrow-derived CXCR4+ SSEA-1+Oct-4+Very Small Embryonic Like (VSEL) stem cells may differentiate into CD45positive long term repopulating hematopoietic stem cells. ASH Meeting:Blood 2007:505.
[40]Jones RJ, Collector MI, Barber JP, et al. Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity. Blood 1996;88:487-91. [PubMed:8695796]
[41]Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature 1990;347:188-189. [PubMed:2395470]
[42]Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med 1996;2:1329-1337. [PubMed:8946831]
[43]Dawn B, TiwariS, Kucia M J, et al. Transplantation of bone Marrow derived very small embryonic2like stem cell s attenuates left vent ricular dysfunction and remodeling after myocardial in farction [J]. Stem Cells,2008,26 (6):164621655.
[44]Zuba2Surma E K, Kucia M, Dawn B, et al. Bone marrow2derived pluripotent very small embryonic2like stem cells (VSELs) are mobilized after acute myocardial infarction [J]. J Mol Cell Cardiol,2008,44 (5):8652873.
[45]Kucia M J, Wysoczynski M, Wu W, et al. Evidence t hat very small embryonic like stem cells are mobilized into peripheral blood [J]. Stem Cells,2008,26 (8):208322092.
[46]Ratajczak M Z, Zuba2Surma E K, Shin D M, et al. Very small embryonic2like (VSEL) stem cells in adult organs and t heir potential role in rejuvenation of tissues and longevity [J]. Exp Gerontol,2008,43 (11):100921017.
[1]Colter D C, Sekiya I, Prockop D J. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cell s in colonies of human marrow stromal cell s [J]. Proc Natl Acad SciUSA,2001,98 (14):7841-7845.
[2]Jiang Y, J ahagirdar B N, Reinhardt R L, et al. Pluripotency of mesenchymal stem cells derived from adult marrow [J]. Nature,2002,418 (6893):41-49.
[3]DpIppolito G, Diabira S, Howard G A, et al. Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential [J]. J Cell Sci 2004,117(14):2971-2981.
[4]Kogler G, Sensken S, Airey J A, et al. A new human somatic stem cell from placental cord blood wit h int rinsic pluripotent differentiation potential [J]. J Exp Med,2004,200 (2):123-135.
[5]Zuba-Surma E K, Kucia M, Ratajczak J, et al."Small stem cells" in adult tissues:Very small embryonic2like stem cells stand up[J]. Cytomet ry A 2008,75A(1):4-13.
[6]Ratajczak M Z, Kucia M, Reca R, et al. Stem cell plasticity re visited: CXCR4-positive cell s expressing mRNA for early muscle, liver and neural cells'hide out'in t he bone marrow [J]. Leukemia,2004,8 (1):29-40.
[7]Kucia M, Ratajczak J, Ratajczak M Z. Are bone marrow stem-cells plastic or heterogeneous that is t he question [J]. Exp Hematol,2005,33 (6):613-623.
[8]Kucia M, Wysoczynski M, Ratajczak J, et al. Identification of very small embryonic like (VSEL) stem cells in bone marrow [J].Cell Tissue Res 2008,331 (1):125-134.
[9]Kucia M, Halasa M, Wysoczynski M, et al. Morphological and molecular characterization of novel population of CXCR4+SSEA-4+Oct-4+very small embryonic like cells purified from human cord blood preliminary report [J] Leukemia,2007,21 (2):297-303.
[10]Halasa M, Baskiewicz Masiuk M, Dabkowska E, et al. An efficient two step method to purify very small embryonic like(VSEL) stem cells from umbilical cord blood (UCB) [J]. Folia Histochem Cytobiol,2008,46 (2):239-243.
[11]Kucia M, Zuba-Surma E K, Wysoczynski M, et al. Adult marrow derived very small embryonic-like stem cells and tissue engineering [J]. Expert Opin Biol Ther,2007,7 (10):1499-1514.
[12]Zuba Surma E K, Kucia M, Wu W, et al. Very small embryonic like stem cells are present in adult murine organs:Image Stream based morphological analysis and distribution studies [J]. Cytom2 et ry A,2008,73A(12):1116-1127.
[13]Ratajczak M Z, Machalinski B, Wojakowski W,et al. A hypothesis for an embryonic origin of pluripotent Oct-4 (+) stem cells in adult bone marrow and other tissues [J]. Leukemia,2007,21(5):860-867.
[14]Kucia M, Wu W, Ratajczak M Z. Bone marrow-derived very small embryonic like stem cells:Their developmental origin and biological significance [J]. DevDyn,2007,236 (12):3309-3320.
[15]Zuba Surma E K, Kucia M, Abdel-Latif A, et al. Morphological characterization of very small embryonic21ike stem cell s (VSELs) by Image Stream system analysis [J]. J Cell Mol Med,2008,12 (1):292-303.
[16]Kucia M, Reca R, Campbell F R, et al. A population of very small embryonic like (VSEL) CXCR4 (+) SSEA-1 (+) Oct-4+stem cells identified in adult bone marrow [J]. Leukemia,2006,20 (5):857-869.
[17]Ratajczak J, Kucia M, Zuba2Surma E, et al. The CD45-Linadult Marrow derived CXCR4+SSEA-1+Oct-4+very small embryonic like (VSEL-SC) stem cells form in vitro spheres which may differentiate into CD45+hematopoietic stem cell s [R]. ASH,48 the annual meeting. Orlando, USA(10):Abstract 280.
[18]Dawn B, TiwariS, Kucia M J, et al. Transplantation of bone Marrow derived very small embryonic-like stem cell s attenuates left vent ricular dysfunction and remodeling after myocardial in farction [J]. Stem Cells,2008,26 (6):1646-1655.
[19]Zuba Surma E K, Kucia M, Dawn B, et al. Bone marrow-derived pluripotent very small embryonic like stem cells (VSELs) are mobilized after acute myocardial infarction [J]. J Mol Cell Cardiol,2008,44 (5):865-873.
[20]Kucia M J, Wysoczynski M, Wu W, et al. Evidence t hat very small embryonic like stem cells are mobilized into peripheral blood [J]. Stem Cells,2008,26 (8):2083-2092.
[21]Ratajczak M Z, Zuba-Surma E K, Shin D M, et al. Very small Embryonic like (VSEL) stem cells in adult organs and t heir potential role in rejuvenation of tissues and longevity [J]. Exp Gerontol,2008,43 (11):1009-1017.
[22]Lamoury F M, Croitoru-Lamoury J, Brew B J. Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct24 and Rex21 [J]. Cytot herapy,2006,8(3):228-242.
[23]Pochampally R R, Smith J R, Ylostalo J, et al. Serum deprivation of human marrow stromal cells (hMSCs) select s for a subpopulation of early progenitor cells with enhanced expression of OCT24 and other embryonic genes [J]. Blood, 2004,103(5):1647-1652.
[24]Zeng L, Rahrmann E, Hu Q, et al. Multipotent adult progenitor cells f rom swine bone marrow [J]. Stem Cells,2006,24 (11):2355-2366.
[25]Pallante B A, Duignan I, Okin D, et al. Bone marrow Oct3/4+cells differentiate into cardiac myocytes via age2dependent paracrine mechanisms [J]. Circ Res,2007,100(1):1-11.
[26]Anjos-Afonso F, Bonnet D. Nonhematopoietic/endot helial SSEA21+cells define t he most primitive progenitors in t he adult murine bone marrow mesenchymal compartment [J]. Blood,2007,109(3):1298-1306.
[27]Ratajczak M Z, Zuba-Surma E K, Machalinski B, et al. Very small embryonic like (VSEL) stem cells:purification f rom adult organs, characterization, and biological significance [J]. StemCell Rev,2008,4 (2):89-99.
[28]Zuba-Surma E K, Wu W, Ratajczak J, et al. Very small embryonic like stem cells in adult tissues2Potential implications for aging[J]. Mech Ageing Dev, 2009,130 (122):58-66.
[29]Kucia M, Zuba-Surma E, Wysoczynski M, et al. Physiological and pathological consequences of identification of very small embryonic like (VSEL) stem cells in adult bone marrow [J]. J Physiol Pharmacol,2006,57 (5):5-18.