The role of fetal-maternal microchimerism as a natural-born healer in integrity improvement of maternal damaged kidney

ABSTRACT Purpose: To identify the fetal stem cell (FSC) response to maternal renal injury with emphasis on renal integrity improvement and Y chromosome detection in damaged maternal kidney. Materials and Methods: Eight non-green fluorescent protein (GFP) transgenic Sprague-Dawley rats were mated with GFP-positive transgenic male rats. Renal damage was induced on the right kidney at gestational day 11. The same procedure was performed in eight non-pregnant rats as control group. Three months after delivery, right ne- phrectomy was performed in order to evaluate the injured kidney. The fresh perfused kidneys were stained with anti-GFP antibody. Polymerase chain reaction (PCR) assay was also performed for the Y chromosome detection. Cell culture was performed to detect the GFP-positive cells. Technetium-99m-DMSA renal scan and single-photon emission computed tomography (SPECT) were performed after renal damage induction and 3 months later to evaluate the improvement of renal integrity. Results: The presence of FSCs was confirmed by immune histochemical staining as well as immunofluorescent imaging of the damaged part. Gradient PCR of female rat purified DNA demonstrated the presence of Y-chromosome in the damaged maternal kidney. Moreover, the culture of kidney cells showed GPF- positive cells by immuno- fluorescence microscopy. The acute renal scar was repaired and the integrity of dam- aged kidney reached to near normal levels in experimental group as shown in DMSA scan. However, no significant improvement was observed in control group. Conclusion: FSC seems to be the main mechanism in repairing of the maternal renal injury during pregnancy as indicated by Y chromosome and GFP-positive cells in the sub-cultured medium.

Large-scale Isolation, Expansion and Characterization of Human Amniotic Epithelial Cells

BACKGROUND AND OBJECTIVES: The human Amniotic epithelial cells (AME) derived from amniotic membrane of placenta have been considered as the potential fetal stem cell source with minimal or no ethical concerns and are important therapeutic tool for anti-fibrotic and regenerative therapies. METHODS AND RESULTS: Here, we evaluated the isolation, media screening, scale-up and characterization of AME cells. The isolation, expansion of AMEs were performed by sequential passaging and growth kinetics studies. The AMEs were characterized using immunocytochemistry, immunophenotyping, In-vitro differentiation, and anti-fibrotic assays. The growth kinetics study revealed that the AME cultured in Ultraculture (UC) and DMEM knockout (DMEM-KO) have prominently higher growth rate compared to others. Overall, the AMEs cultured from 5 different media retained basic morphological characteristics and the functional characteristics. CONCLUSIONS: Our result suggests that the AMEs can be successfully cultured in UC based complete media without losing its epithelial cell characteristics even after passaging for passage 2 (P2). However, a careful and methodical pre-clinical and clinical translation studies need to be conducted to show its safety and efficacy.

Isolation, culture, characterization and cryopreservation of stem cells derived from amniotic mesenchymal layer and umbilical cord tissue of bovine fetuses / Isolamento, cultura, caracterização e criopreservação de células tronco derivadas de camada amniótica mesenquimal e de tecido do cordão umbilical de fetuses bovinos

Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.(AU)

As células tronco mesenquimais (CTMs) estão presentes na maioria dos tecidos adultos e possuem grande capacidade de multiplicação. Quando cultivadas in vitro são capazes de se auto renovar e dar origem a novos tipos celulares. As células tronco mais utilizadas, tanto na medicina humana como na medicina veterinária são as células tronco mesenquimais derivadas da medula óssea e do tecido adiposo. Atualmente, uma grande tendência para a utilização de CTMs obtidas de tecidos fetais, como a membrana amniótica (MA), matriz extravascular do cordão umbilical (TCU) e sangue do cordão umbilical (SCU) pode ser observada, já que estas fontes podem ser colhidas no momento do parto por uma técnica não invasiva. Dessa forma, o objetivo deste estudo foi isolar, caracterizar, diferenciar e criopreservar CTMs obtidas de MA e TCU de fetos bovinos colhidos no momento do parto e de fetos do terço inicial da gestação em abatedouro-frigorífico. As células foram recuperadas por meio de digestão enzimática tecidual, realizada com solução de colagenase 0,1%. Foram colhidas amostras de MA e TCU no momento do parto (n=6) e de MA e TCU no terço inicial de gestação (n=6), as quais foram submetidas às análises morfológicas, imunofenotípica por imunocitoquímica e citometria de fluxo, diferenciações in vitro nas linhagens osteogênica, adipogênica e condrogênica e ainda, avaliação da viabilidade após a criopreservação por citometria de fluxo. Todas as amostras dos diferentes grupos demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico todas as amostras foram imunomarcadas para CD44, NANOG e Oct-4, com ausência de marcação para MHC II. Na análise imunofenotipica por citometria de fluxo, todas as amostras apresentaram marcação para CD44, ausência de marcação para ou baixíssima expressão de CD34 (MA: TI-0,3%a, PA-3.4%b; TCU: TI-0,4%, PA-1.4%) e nula ou baixa expressão de MHC II (MA: TI-1.5%a, PA-9.7%b; UCT: TI-0.7%a, PA-5.7%b. Apresentaram também capacidade de diferenciação in vitro nas três linhagens mesodermais e quando analisadas pós criopreservação por citometria de fluxo, todas as amostras apresentaram viabilidade de 80%. Estes resultados indicam que MA e TCU, obtidos tanto no momento de parto como em abatedouro, de fetos bovinos podem ser utilizados como fonte não invasiva e indolor de CTMs e possibilitam a formação de bancos de armazenamento de células.(AU)

Avances en la medicina reconstructiva: células madre y células madre inducidas / Advances in reconstructive medicine: stem cells and induced stem cells

La utilización de células indiferenciadas embrionarias y de células diferenciadas inducidas para que se comporten como las anteriores permite dar origen adiferentes tejidos que pueden ser usados en medicina reconstructiva en reemplazo de los deteriorados...

Effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor cells MC3T3-E1 / 浙江大学学报·医学版

To investigate the effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor (MC3T3-E1) cells.miR-705 mimics, inhibitors and negative control were transfected into MC3T3-E1 cells. Alkaline phosphates (ALP) staining were performed and quantified after 7 days of osteogenic medium induction. The mRNA and protein expression levels of runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected by real-time RT-PCR and Western blot after 14 days of osteogenic induction. Alizarin red staining was performed and quantified in MC3T3-E1 cells after 21 days of osteogenic induction.After 7 days of osteogenic induction, ALP staining showed that overexpression of miR-705 significantly reduced ALP activity, whereas knockdown of miR-705 increased ALP activity (all<0.05). Consistently, after 14 days of osteogenic induction, mRNA and protein expressions of Runx2 and OCN were suppressed by overexpression of miR-705, whereas they were promoted by knockdown of miR-705 (all<0.05). After 21 days of osteogenic induction, alizarin red staining showed that overexpression of miR-705 significantly reduced the formation of mineralized node, the opposite results were found in miR-705 knockdown group (all<0.05).miR-705 can inhibit the osteogenic differentiation of MC3T3-E1 cells.

Cell-based therapy for kidney disease

The prevalence of renal disease continues to increase worldwide. When normal kidney is injured, the damaged renal tissue undergoes pathological and physiological events that lead to acute and chronic kidney diseases, which frequently progress to end stage renal failure. Current treatment of these renal pathologies includes dialysis, which is incapable of restoring full renal function. To address this issue, cell-based therapy has become a potential therapeutic option to treat renal pathologies. Recent development in cell therapy has demonstrated promising therapeutic outcomes, in terms of restoration of renal structure and function impaired by renal disease. This review focuses on the cell therapy approaches for the treatment of kidney diseases, including various cell sources used, as well recent advances made in preclinical and clinical studies.

Establishment of High Throughput Screening System Using Human Umbilical Cord-derived Mesenchymal Stem Cells

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cordderived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.

O intervalo de tempo entre coleta e processamento do sangue de cordão umbilical influencia na qualidade da amostra? / Does the time between collecting and processing umbilical cord bloodsamples affect the quality of the sample?

Objective: To assess the association between the time from umbilical cord blood collection until processing and the quality of the sample. Methods: Umbilical cord blood samples collected during the third stage of labor were placed in temperature-controlled boxes for the transport of biological material and sent to an umbilical cord blood bank, where the number of nucleated cells, viable cells and CD34+ cells were counted, and samples were additionally tested for contamination at the following time intervals: up to 24 hours, up to 48 hours and up to 72 hours following sampling. Data were analyzed using the multivariate analysis of variance (MANOVA) and compared using McNemar's X2 test. Significance was defined at p < 0.05. Results: Means and medians of the number of nucleated cells, viable cells and CD34+ cells decreased significantly (p < 0.0001) as a function of the increased time between sampling and analysis, the difference between 24 and 48 hours being less than the difference between 24 and 72 hours. A linear correlation was found between the mean number of viable cells and CD34+ cells at the three moments of analysis. Contamination testing was negative in all samples. Conclusion: The increase in time interval from sampling until analysis negatively affected the number of nucleated cells, viable cells and CD34+ cells but was not associated with specimen contamination. A linear correlation was found between decrease in the number of viable cells and CD34+ cells.

Objetivo: Avaliar a associação do intervalo de tempo entre coleta e processamento do sangue de cordão umbilical e a qualidade da amostra. Métodos: As amostras de sangue de cordão umbilical, colhidas no terceiro período do parto, foram acondicionadas em caixas homologadas para transporte de material biológico, com monitoração da temperatura, e enviadas a um banco de sangue de cordão umbilical, onde foram submetidas à contagem do número de células nucleadas, do número de células viáveis, do número de células CD 34+ e pesquisa de contaminação, nos intervalos de tempo de até 24, até 48 e até 72 horas. Os dados foram analisados pelo teste de variância para medidas repetidas MANOVA e comparados por meio do teste do X2 de Mc Nemar, considerando-se nível de significância de 5%. Resultados: As médias e as medianas do número de células nucleadas, número de células viáveis e número de células CD34+ tiveram quedas significativas (p < 0,0001) com o aumento do intervalo de tempo de coleta/processamento, sendo entre 24 e 48 horas menor do que a comparação entre 24 e 72 horas. Constatada correlação linear entre as médias de células viáveis e células CD34+ nos três momentos da análise. A pesquisa de contaminação foi negativa em todas as amostras. Conclusão: O aumento do intervalo de tempo de coleta/processamento influenciou negativamente na contagem de células nucleadas, células viáveis e CD34+ e não esteve associado à contaminação das amostras. Foi constatada correlação linear entre a queda do número de células viáveis e de células CD34+.

Viabilidade de células do sistema nervoso central fetal no tratamento da lesão medular em ratos / Viability of fetal central nervous system cells in the treatment of spinal cord injury in rats

OBJETIVOS: Propor um modelo experimental de transplante de células do sistema nervoso fetal de ratos Wistar para o sítio de lesão medular de ratos adultos que permitisse sua sobrevivência e integração para possibilitar protocolos de pesquisa que identificarão outros fatores de regeneração e recuperação funcional pós trauma raquimedular. MÉTODOS: Vinte ratos adultos foram submetidos a laminectomia, e lesão de 5mm de hemimedula realizada com auxílio de microscópio óptico. Quinze deste ratos tiveram seu sítio de lesão medular transplantado com células do sistema nervoso central de fetos de rato; os ratos foram monitorados por 2 dias e tiveram sua coluna vertebral extraída para análise histológica. RESULTADOS: Evidenciou-se que em 60 por cento dos casos as células transplantadas permaneciam viáveis no sítio da lesão e que a reação inflamatória no grupo transplantado era sempre maior que no grupo controle. CONCLUSÃO: O presente trabalho demonstrou a possibilidade de contar com o modelo de pesquisa para transplante de células fetais que permanecem viáveis 2 dias após seu implante.

OBJECTIVE: To propose an experimental model for transplantation of fetal cells from the nervous system of Wistar rats to the site of spinal cord injury in adult rats, to enable their survival and integration for research protocols that identify other factors of regeneration and functional recovery following spinal cord trauma. METHODS: Twenty adult rats were submitted to laminectomy and a 5mm incision was made, using an optical microscope, In fifteen of these rats, the site of the spinal cord lesion was transplanted with cells from the fetal rat central nervous system; the rats were monitored for two days, then the spinal cord was removed for histological analysis. RESULTS: In 60 percent of cases, the transplanted cells remained viable in the site of the lesion; the inflammatory response in the transplanted group was always greater than in the control group. CONCLUSION: This study demonstrates the potential use of this research model for use in the transplantation of fetal cells that remain viable two days after their implantation.

Investigation of polyanhydride-glucan as stem cell carriers / 生物医学工程学杂志

<p><b>UNLABELLED</b>The purpose of this study with regard to the effects of polyanhydride--three-dimensional vector-glucan material on the fetal liver stem cell adhesion and proliferation was to find a new carrier. The methods of two-step collagenase perfusion digestion and liquid Percoll discontinuous density gradient centrifugation were used for the separation of fetal liver stem cells. The fetal liver stem cells were selected and cultivated in the polyanhydride-three-dimensional vector-glucan material. Inverted microscope was used to observe cell adhesion and growth status. Also performed were: Calculation of the rate of cell adhesion; MTT assay of the cells in each group absorbance value (OD value); collecting and counting the cells on the carrier scaffold. Then the cell carriers histological sections (HE staining) were observed. On the 7th day of cell culture, the cells were subjected to immunofluorescence staining and flow cytometry.</p><p><b>RESULTS</b>polyanhydride-three-dimensional vector-glucan promoted liver stem cells growth and adhesion. There were active functions of the liver stem cells within carrier materials. In the three-dimensional surface and the internal culture of liver stem cell, proliferation was sustained. After 40 days, the polyanhydride co-culture-three-dimensional vector-glucan showed no sign of toxicity to stem cells. Human fetal liver stem cells attached to the polyanhydride--three-dimensional vector-glucan stent. The cell proliferation went on well and exhibited sustained expression of markers; 7 days training led up to an increase of 19.7 percent in the number of cells. Conclusively, polyanhydride-three-dimensional vector-glucan can be used for promoting the proliferation of liver stem cells, and liver stem cells can be used as vectors in liver tissue engineering.</p>

Anti-aging effect of transplantation of mouse fetus-derived mesenchymal stem cells / 生理学报

To determine the role of allogeneil graft of mesenchymal stem cells in mammalian longevity, mesenchymal stem cells were isolated from BALB/c mouse uterine-incision delivery fetus by two successive density gradient centrifugations, and then were purified and amplified by adherent culture. Identified P1 mesenchymal stem cells were injected (i.v.) through vena caudalis into the 15-month-old female BALB/c mice three times. The mice were evaluated with ultrasoundcardiogram, autopsy, score of cardiac, skin, lung, kidney, colon histopathology and serum total superoxide dismutase activity, maleic dialdehyde content, glutathione peroxidase activity. The results showed that after transplantation, the long-term surviving stem cells were found to be located in many organ tissues with in situ Y chromosomal hybridization dyeing. Median life span was increased in these animals after transplantation. Skin, cardiac, lung, kidney and colon pathology development were delayed. The retrogradation of heart function was attenuated, the increase of heart mass index (the mass of heart/the mass of the body), and serum maleic dialdehyde content, the decrease of spleen mass index (the mass of spleen/the mass of the body), serum total superoxide dismutase activity and glutathione peroxidase activity were reduced three months after transplantation (all P<0.05). These results support the idea that longevity can be enhanced by transplantation of mesenchymal stem cells and reinforce the hypothesis of mesenchymal stem cell as antiager.

Growth factor-expressing human neural progenitor cell grafts protect motor neurons but do not ameliorate motor performance and survival in ALS mice

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD(1G93A)) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.

Hallazgos histológicoultraestructurales característicos de las células madres (stem cell embriónico fetales): primer reporte internacional / Ultra structural histologic findings characteristic of embryonic fetal stem cells: first international report

Se buscó la evidencia de crecimiento muscular o biogénesis que debería haberse iniciado a partir del transplante de Stem cell embriónico fetales de donantes con el propósito de observar los dos tipos de ADN distintos tanto del receptor como del donante. Materiales y métodos: esta investigación se realizó luego de recibir fragmentos de músculo cardiaco del ventrículo izquierdo en los cuales se implantaron dichas células directamente en el miocardio mediante 80 inyecciones de 1ml cada una en todas las paredes de ambos ventrículos, con especial énfasis en el izquierdo. Las biopsias fueron fijadas con glutaraldehido al 3, post-fijazas con Tetraóxido de Osmio al 1, deshidratadas en diversos gradientes ascendentes de alcohol etílico, preembedidas en Oxido de propileno, embebidas en resina Spurr para realizar los bloques, los cuales fueron cortados por ultramicrotomía, teñidas con acetato de uranilo y citrato de plomo, y finalmente observadas al Microscopio Electrónico de Transmisión JEOL- JEM 1010. Resultados: lo más relevante en esta investigación son los hallazgos encontrados en las mitocondrias que se presentaban hipertróficas, redondeadas con crestas vesiculares, siendo lo distintivo y el más importante hallazgo la presencia de una banda recta trilaminar con una estructura central electrondensa proteica, la misma que atraviesa perpendicularmente en toda la longitud de la mitocondria, formando una “Barra germinativa pluripotencial mitocondrial”, hallazgo propio distintivo de las células Madre. Conclusiones: siendo en la mitocondria esta inusual barra recta transversal electrondensa un importante aporte científico distintivo que identificaría plenamente a las células transplantadas del tipo de Stem cell embriónico fetales, diferentes a las mitocondrias con crestas cortas irregulares onduladas paralelas vacías, dando con esto una diferenciación característica que determinaría su capacidad de ser pluripotencial y de autorenovación.

We looked up an evidence of muscle growth or biogenesis that should have started from the transplant of embryonic fetal stem cells from donors in order to observe the two distinct AND types of both, the receptor and the donor. Materials and methods: this research was made after receiving fragments of the left ventricle cardiac muscle where those cells were implanted directly in the myocardio by means of 80 injections of 1 ml each in all the walls of both ventricles, with special stress in the left one. Biopsies were fixed with 3 glutaraldehido, post fixed with 1 Osmio Tetraoxide, dehydrated in different ascending gradients of ethyl alcohol, pre soaked in propylene oxide, soaked in Spurr resin to make the blocks which were cut by ultramicrotomy, dyed with uramil acetate and lead citrate, and finally observed at the JEOL – JEM 1010 Transmission Electronic Microscope. Results: the most outstanding facts in this research are the findings met in the mitochondrion that were hypertrophic, rounded with vesicular crests, and what makes difference and the most important finding is the presence of a tri-laminar straight strip with a central proteinic electrodense structure which crosses perpendicularly the mitochondrion in its whole length, giving form to a “Mitochondrial pluripotential germinative Bar” distinctive finding of stem cells. Conclusions: this unusual electrodense, transverse, straight bar is an important scientific contribution and a distinctive that would identify completely the transplanted stem cells embryonic fetal type, different from mitochondrion with short irregular wavy, parallel, empty crests, giving this way a characteristic differentiation that would determine their capability of being pluripotential and with self-renovation.

[Assessment of capability of fetal and adult neural stem cells to differentiate into endothelial cells]

Neural stem cells exist in various regions of the developing and adult central nervous system [CNS]. They are undifferentiated cells, capable of both self-renewal and producing neurons and glial cells. In addition to generating different types of neural cells, NSCs are capable of producing cells of other tissues. In this study NSCs were isolated from mouse brain and their capability of differentiation into endothelial cells was evaluated. Neural stem cells [NSCs] were isolated from lateral wall of the lateral ventricle of the adult and fetal mouse brain and cultured in serum-free DMEM-F12 medium in the presence of basic fibroblast growth factor [bFGF], epidermal growth factor [EGF] and B27 supplement. Neurospheres were plated on the fibronectin coated culture slides. DMEM-F12 media supplemented with 10% FBS was used for differentiation of NSCs into endothelial cells. Differentiated cells were evaluated by Immuno-histochemistery, RT-PCR and tube formation assays. The results of this study revealed culture of fetal neural stem cells in fibronectin coated and also DMEM-F12 media containing 10% FRS led to differentiation of NSCs into endothelial cells. When differentiated cells were transferred into extra cellular matrix or matrigel, they produced capillary like structures characteristic of endothelial cells. The results of immuno-histo-chemistry and RT-PCR showed differentiated cells can absorb low density lipoproteins and express CD31, VE, cadherin and Flk-1 genes, and also can adhere to BS-1lectin. Unlike neural stem cells isolated from mouse fetus, the isolated cells from adult mouse brain did not differentiate into endothelial cells.This study reveals neural stem cells isolated from fetal mouse brain and adult mouse brain do not show similar behavior in differentiating into endothelial cells

D2 receptor expression on immortalized human neural progenitor cell line hNPC-TERT in vitro and in vivo / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe D(2) receptor expression on human neural progenitor cell line hNPC-TERT before and after transplantation into rabbit central nervous system.</p><p><b>METHODS</b>D(2) receptor expression on cultured hNPC-TERT cells was verified and quantitatively analyzed with immunofluorescence assay and receptor radio ligand binding assay, respectively. 3 x 10(6) hNPC-TERT cells were implanted in the spinal cord of New Zealand rabbit with HeLa cells as the control. Two days after implantation, positron-emission tomography (PET) scan with (11)C-raclopride as the radiotracer was performed in the living animals or for the isolated spinal cords, and cryosections of the spinal cord containing the implanted cells were prepared for immunofluorescence assay.</p><p><b>RESULTS</b>Cultured hNPC-TERT cells showed high expression of D(2) receptor (Bmax=8 x 10(4)). PET scans of the rabbits identified visible radioactive accumulations at the site where hNPC-TERT cells were implanted but not at the site of HeLa cell implantation. Region of interest analysis showed a significant difference between the two cells in the maximal standard uptake value at the cell implantation sites. The results were further confirmed with ex vivo PET imaging of the spinal cord and tissue immunofluorescence assay.</p><p><b>CONCLUSION</b>Human neural progenitor cells hNPC-TERT highly express dopamine D(2) receptors and retain this capacity after implantation into the spinal cord, suggesting their potential for treatment of such nerve system disease as Parkinson syndrome.</p>

Intrasplenic heterotransplantation of in vitro cultured human fetal hepatic stem cells for treatment of acute liver injury in mice with severe combined immunodeficiency / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the in vivo colonization, migration, and differentiation of in vitro cultured human fetal hepatic stem cells (HSCs) following intrasplenic transplantation for treatment of acute liver injury in mice with severe combined immunodeficiency (SCID).</p><p><b>METHODS</b>Human fetal HSCs were isolated from the normal fetal liver (16-24 weeks) and purified, and the morphology of HSCs was observed under optical and transmission electron microscopes. The expressions of stem cell markers were examined in these HSCs by means of immunocytochemistry and flow cytometry. The passaged human fetal HSC suspension (0.2 ml) were injected into the spleen of SCID mice with acute liver injury induced by two-third partial hepatectomy, and 15, 30, 60, and 90 days after cell transplantation, immunohistochemistry was performed to examine the location and expressions of human hepatocytes, alpha1-AT and AFP antigen in the spleen and liver of the recipient SCID mice. PAS staining was used to examine the expression of glycogen and RT-PCR employed for detection of the expressions of AFP and albumin mRNA in the spleen of the mice on the scheduled time points.</p><p><b>RESULTS</b>Under optical microscope and transmission electron microscope, most of the HSCs were small, about 1/6 to 1/3 of the size of the hepatocyte, with relatively large nucleus-cytoplasm ratio and only small quantities of endocytoplasmic reticulum, chondriosome, and ribosome. Immunohistochemistry and flow cytometry identified positive expressions of AFP, Thy-1, C-kit, CD34 and CK19 in the HSCs, and after cell transplantation, positive expressions of human hepatocyte, alpha1-AT, and AFP antigen occurred in the liver and spleen of the recipient SCID mice. PAS staining confirmed the presence of glycogenosome in the spleen of the mice following cell transplantation. RT-PCR on days 30, 60, and 90 showed positive expressions of human AFP and albumin mRNA in the spleen of the mice.</p><p><b>CONCLUSION</b>Human fetal HSCs can survive and settle in the spleen and liver, and migrate to the damaged liver of the recipient mice after intrasplenic transplantation, with the capacity of proliferation and differentiation into hepatocytes in the recipient target organs.</p>

How to improve the survival of the fetal ventral mesencephalic cell transplanted in Parkinson's disease? / 神经科学通报·英文版

It has been extensively confirmed that fetal ventral mesencephalic cell (VMC) transplantation can ameliorate the symptoms of Parkinson's disease (PD). But there are still several problems to be resolved before the extensive clinical application of this technology. The major limitations are the poor survival of grafted dopamine (DA) neurons and restricted dopaminergic reinnervation of host striatum. Some attempts have been made to solve these problems including use of some trophic factor and co-transplantation with neural/paraneural origins. The purpose of this review is to overview advances of the means improving the survival of grafts and their current limitations.

Primary study on the culture of human fetal follicle bulge cells and their differentiation into sebaceous gland / 中华烧伤杂志

<p><b>OBJECTIVE</b>To develop a rapid and reproducible method for the culture of human fetal hair follicle bulge cells, and observe the plasticity of its differentiation into sebaceous gland in vitro.</p><p><b>METHODS</b>The bulge cells isolated from fetal human hair follicles by enzymatic digestion (digestion method) and manual microdissection (conventional method) were cultured and passaged respectively, the efficiency and biological features of cells were investigated , the clone forming efficiency was assayed by MTT, and the expression of K19 was further compared by immunocytochemistry (ABC). The morphological change and the expression of EMA of bulge cells were also observed after induction.</p><p><b>RESULTS</b>By conventional method, 8-10 bulges were harvested in one hour, 40%-50% of their cells were found to adhere to the culture plate after culturing for 48h, and they became confluent after 14 days. In comparison, about 100 bulges were harvested in one hour by digestion method, the adherence efficiency of their cells was 30% after cultivation for 12h and became confluent after 7 days. The cells grew larger with time, with irregular shape and droplets of lipid around the nucleus. The clone forming efficiency of bulge cells cultured by digestion method was (18.2 +/- 2.1) %, which was much higher than that of cells obtained by conventional method[ (12.7 +/- 3.4) %, P < 0.05]. Immunocytochemistry staining showed that positive staining of K19 was observed in most of the bulge cells, with a large amount of brown granules in the cytoplasm.</p><p><b>CONCLUSION</b>Human hair follicle bulge cells can be efficiently cultured and multiplied in vitro, and they retained the characteristics of stem cells. And they have the potential to differentiate into sebaceous glands by induction in vitro.</p>

Construction of fusion expression vector EGFP-PDX-1 and its transfection into rat fetal hepatic stem cells by electroporation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the fusion expression vector of pancreatic-duodenal homeobox gene 1 (PDX-1) fused to green fluorescent protein (GFP) capable of stable expression in fetal rat hepatic stem cells after transfection by electroporation.</p><p><b>METHODS</b>PDX-1 cDNA was amplified from SK900/BLSCRIPT plasmid and cloned into the multiple cloning site of pEGFP-C1 to obtain the recombined plasmid pEGFP-C1-PDX-1. Rat fetal hepatic stem cells were isolated, cultured, identified and transfected with the recombinant vector by electroporation, followed by observation of these cells with fluorescent microscope. The result of transfection was analyzed by RT-PCR and cell growth curve.</p><p><b>RESULTS</b>Identification by enzyme digestion confirmed successful construction of the recombinant vector. Fetal hepatic stem cells can stably express GFP and PDX-1 for a period of time, and their growth and proliferation was not obviously affected after transfection.</p><p><b>CONCLUSION</b>The fusion expression vector of EGFP-PDX-1 is successfully constructed and stably expressed in rat fetal hepatic stem cells, which may facilitate the study of the role of PDX-1 in stem cell differentiation into insulin-producing cells.</p>

Experimental study on growth, proliferation and differentiation of neural stem cell from subventricular zone of human fetal brain at different gestational age / 中华儿科杂志

<p><b>OBJECTIVE</b>To study growth characteristics of neural stem cells (NSCs) from subventricular zone (SVZ) of the different human fetal brain at different gestational age and to provide experimental and theoretical evidences for clinical application of NSCs for treatment of certain diseases.</p><p><b>METHODS</b>Ninety human embryos at gestational age 16 - 36 weeks were collected and were divided into six groups according to gestational age: 16 w, 20 w, 24 w, 28 w, 32 w and 36 w. Each group had 15 embryos and brain tissues were taken from each embryo's SVZ. All subjects had congenital heart disease or digestive tract abnormity diagnosed with B ultrasound at antepartum, but none had abnormal development of brain. Pregnant mother and her husband desire termination of pregnancy. The morphology, existing mode and the number of neural stem cells in subventricular zone were examined with immunohistochemical method. The NSCs in subventricular zone were cultured, passaged and differentiated with cell culture technique, then were identified with immunohistochemical method.</p><p><b>RESULTS</b>NSCs in SVZ from the different human fetal brain existed in a scattered manner in the network formed by stellate cells, NSCs had round, ellipse and fusiform shape, especially in stellate shape. NSCs had larger and smaller size and distributed in dense or scattered forms, each having zero to two enations, most had one or two. NSCs had less cytoplasm. The nucli of the NSCs had a round shape with loose chromatin and 1 - 4 nucleoli. Most of NSCs existed in singular scattered form, some of them showed symmetrical or asymmetrical division, some of them showed synaptic connection with other NSCs. The number of NSCs in SVZ from groups with different fetal age decreased with increasing gestational age (chi(2) = 4644.602, P < 0.01). NSCs in SVZ from the different human fetal brain cultured with serum-free medium formed typical neurospheres in suspension. The cells could be passaged continuously, and could express nestin antigen. Serum-contained medium induced neural stem cells to differentiate and express specific antigens of neuron, astrocyte and oligodendrocyte.</p><p><b>CONCLUSIONS</b>NSCs existed in SVZ of human embryos at different gestational age. There are differences in morphology, existing pattern and the number of NSCs in SVZ at different gestational age. NSCs in SVZ at different gestational age may be cultured in vitro.</p>