Skin-derived precursors (SKPs) have potential to differentiate to various cell types including osteoblasts, adipocytes and neurons. SKPs are a candidate for cell-based therapy since they are easily accessible and have multipotency. Most mammalian cells are exposed to a low oxygen environment with 1 to 5% O2 concentration in vivo, while 21% O2 concentration is common in in vitro culture. The difference between in vitro and in vivo O2 concentration may affect to the behavior of cultured cells. In this report, we investigated the effect of hypoxic condition on stemness and proliferation of SKPs. The results indicated that SKPs exposed to hypoxic condition for 5 days showed no change in proliferation. In terms of mRNA expression, hypoxia maintained expression of stemness markers; whereas, oncogenes, such as Klf4 and c-Myc, were downregulated, and the expression of Nestin, related to cancer migration, was also downregulated. Thus, SKPs cultured in hypoxia may reduce the risk of cancer in SKP cell-based therapy.
Adipocytes ; Anoxia ; Cells, Cultured ; In Vitro Techniques ; Nestin ; Neurons ; Oncogenes ; Osteoblasts ; Oxygen ; RNA, Messenger

OBJECTIVE: This study is aimed whether bcl-xl could protect C4 cells from the cell death induced by serum deprivation. METHODS: The transient transfection of the bcl-xl gene was made with a LipofectAMINE reagent. An immunohistocytochemical assay and Western-blotting were performed to examine the bcl-xl transfection into the C4 cells. In order to analyze the effect of the bcl-xl transfection, the number of cells on the well plate were serially counted each day, for 5 days, from the 2nd to the 6th day after transfection. The number of GFP-positive cells in the defined fields, following serum deprivation, was counted using fluorescence microscopy, and the total number of viable cells, including transfected cells, were also assessed. RESULTS: Immunocytochemical staining showed positive cells in 52% of nestin staining, 60% of GFAP and 20% of MAP-2. The number of cells decreased after transfection using the LipofectAMINE in the serum free medium (p<0.001). Western blotting using an anti-human bcl-xl antibodies showed that bcl-xl was expressed in both the non-transfected and bcl-xl transfected C4 cells. Cell death in the C4 cells, and the number of cells, were serially monitored each day for 5 days. In the bcl-xl transfected cells, the cell death induced by serum deprivation was significantly inhibited and delayed compared to that in the control cells (p<0.001). CONCLUSION: It is suggested that the bcl-xl transfected human neural progenitor cells might improve the survival of the grafted cells, and may be an alternative source of cells for neural transplantation in degenerative diseases.
Antibodies ; Blotting, Western ; Cell Death ; Humans* ; Microscopy, Fluorescence ; Nestin ; Stem Cells* ; Transfection ; Transplants

OBJECTIVETo explore the role of microtubule-associated protein 2 (MAP-2) and nestin in the development of tongue muscles of human embryos and fetuses.

METHODSPV immunohistochemistry was used to detect the expressions of MAP-2 and nestin proteins in the tongue tissues of human embryos and fetuses at the second, third and fourth months of gestation.

RESULTSMAP-2 and nestin positivity was detected in the tongue muscles of human embryos at 2 to 4 months of gestation. In the embryos at the second month of gestation, no obvious MAP-2 positive cells were found in the tongue muscles; at 3 and 4 months, the number of MAP-2-positive cells in the tongue muscles was 24.14∓8.28 and 15.86∓3.89, with the expression intensity of 109.42∓11.62 and 124.27∓8.73, respectively. At 2, 3 and 4 months of gestation, the number of nestin-positive cells in the tongue muscles was 12.50∓3.17, 19.00∓7.63, and 22.80∓6.91, with expression intensity of 119.99∓24.02, 102.20∓11.76, and 98.24∓10.66, respectively. As the gestational age increased, the number of MAP-2-positive cell number continued to decline following a transient increase but the expression intensity kept increasing; nestin-positive cells increased continuously but the expression intensity kept decreasing in the embryonic or fetal tongue muscles.

CONCLUSIONMAP-2 and nestin proteins are involved in the regulation of the development of tongue muscles in human embryos and fetuses.

Humans ; Microtubule-Associated Proteins ; metabolism ; Muscle, Skeletal ; embryology ; Nestin ; metabolism ; Tongue ; embryology

Astrocytoma ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; In Vitro Techniques ; Nestin ; metabolism

OBJECTIVETo observe the morphological changes during development of the ventricular zone (VZ) and subventricular zone (SVZ) of human fetus and the distribution pattern of neural stem cells in the VA and SVZ.

METHODSHuman fetuses at the gestational ages of 9-11 weeks, 14-16 weeks, 22-24 weeks and 32-36 weeks were collected, and the brain sections of the VZ/SVZ under the frontal lobe were examined for cytoarchitecture and distribution of nestin-positive cells with HE staining, immunohistochemistry or immunofluorescence.

RESULTSThe thickness of VZ underwent no significant changes at the gestational ages of 9-24 weeks (P>0.05) and became obviously thinner at 32 weeks (P<0.05), while the thickness of SVZ increased during 9-24 weeks (P<0.05) without obvious thinning at 32 weeks (P>0.05). VZ was thicker than SVZ at 9-11 weeks but became markedly thinner than SVZ after 14 weeks (P<0.05). The VZ contained denser cells than SVZ and showed a distinct boundary between the VZ and SVZ. Large numbers of nestin-positive cells were detected in the VZ and SVZ, and nestin immunoreactivity was found primarily in the cell processes and occasionally in the soma. Some nestin-positive cells in the SVZ had 1-3 processes. Nestin immunoreactivity in the VZ and SVZ gradually grew weak with development. The cells positive for both nestin and Ki67 were located mainly in the inner zone of the VZ and throughout the SVZ, where some nestin-positive but Ki67-negative cells were also found.

CONCLUSIONThe SVZ fully extends and the neural stem cells in the VZ/SVZ can be morphologically heterogeneous during the development of fetal human brain.

Fetus ; Frontal Lobe ; cytology ; embryology ; metabolism ; Humans ; Nestin ; metabolism ; Neural Stem Cells ; metabolism ; Neurons ; metabolism

OBJECTIVETo explore the role of microtubule-associated protein 2 (MAP-2) and nestin in the development of tongue muscles of human embryos and fetuses.

METHODSPV immunohistochemistry was used to detect the expressions of MAP-2 and nestin proteins in the tongue tissues of human embryos and fetuses at the second, third and fourth months of gestation.

RESULTSMAP-2 and nestin positivity was detected in the tongue muscles of human embryos at 2 to 4 months of gestation. In the embryos at the second month of gestation, no obvious MAP-2 positive cells were found in the tongue muscles; at 3 and 4 months, the number of MAP-2-positive cells in the tongue muscles was 24.14∓8.28 and 15.86∓3.89, with the expression intensity of 109.42∓11.62 and 124.27∓8.73, respectively. At 2, 3 and 4 months of gestation, the number of nestin-positive cells in the tongue muscles was 12.50∓3.17, 19.00∓7.63, and 22.80∓6.91, with expression intensity of 119.99∓24.02, 102.20∓11.76, and 98.24∓10.66, respectively. As the gestational age increased, the number of MAP-2-positive cell number continued to decline following a transient increase but the expression intensity kept increasing; nestin-positive cells increased continuously but the expression intensity kept decreasing in the embryonic or fetal tongue muscles.

CONCLUSIONMAP-2 and nestin proteins are involved in the regulation of the development of tongue muscles in human embryos and fetuses.

Fetus ; metabolism ; Humans ; Microtubule-Associated Proteins ; metabolism ; Nestin ; metabolism ; Tongue ; embryology ; growth & development ; metabolism

Recently, there has been considerable attention focused on the multipotent progenitor cells existing in ependymal and subependymal layer. However, almost all results have been derived from brain or injured CNS researches. So, the studies on the developmental characteristics of intact spinal ependymal layer have been relatively ignored. In the present study, we labeled rat spinal cord with nestin, bromodeoxyuridine (BrdU), and glial fibrillary acidic protein (GFAP) antibodies in order to track the differentiation and proliferative capacity of rat ependymal layer cells according to their developmental stages. At embryonic day 14 (E14), a number of cells in the spinal ependymal layer, especially constituting the alar and basal plates, showed extensive nestin immunoreactivities (ir). They also showed active proliferative capacities, because many nuclei of nestin-ir cells were also BrdU-ir. From postnatal day 0 (P0), nestin-ir cells were almost completely disappeared, and from P7, no nestin-ir cells could be detected. However, BrdU-ir nuclei continued to be identified until P14. These results suggested that the cells in the spinal ependymal layer retain their proliferative capacity until later stage of development. On the other hand, no GFAP-ir cells could be identified in the ependymal layer in our experimental period.
Animals ; Antibodies ; Brain ; Bromodeoxyuridine* ; Glial Fibrillary Acidic Protein ; Hand ; Nestin* ; Rats* ; Spinal Cord ; Stem Cells

Nestin, a type VI intermediate filament,is a marker for stem cells.Although it is expressed abundantly in various organs during development,its expression in adults is restricted to certain types of cells.However,nestin is reinduced in activated cells involved in the regeneration and survival of injured tissues.We investigated the expression of nestin in the ischemia-reperfusion injured rat kidney by immunohistochemistry using anti-nestin antiserum. Kidneys were preserved from normal adults and at 1,3,5,7 and 14 d after ischemia-reperfusion injury,and processed using pre-embedding.In the normal adult kidney,nestin was expressed strongly in the glomerular podocyte.Capillary endothelial cells,except for those of the glomeruli,showed nestin positivity.Fibroblast-like interstitial cells were also nestin positive except for lipid-laden interstitial cells of the inner medulla.After ischemia-reperfusion injury,the renal expression of nestin increased progressively to 7 d and then returned almost to a normal level by 14 d.These changes of nestin expression were attributed mainly to changes in the number and staining intensity of immunostained interstitial cells.The podocytes and endothelial cells showed no change in immunoreactivity throughout any stage in the experimental animals.Interestingly,nestin-positive tubular cells,which were nestin negative in normal kidney,were observed from 3 to 14 d.Nestin immunostained cells were increased in the interstitium around these nestin-positive tubules.These results suggest that nestin is induced in both interstitial cells and regenerating tubular cells and that it can be used as a histological marker related to epithelial-mesenchymal transformation in the injured kidney.
Adult ; Animals ; Endothelial Cells ; Epithelial-Mesenchymal Transition ; Humans ; Immunohistochemistry ; Kidney* ; Nestin* ; Podocytes ; Rats* ; Regeneration ; Reperfusion Injury*

Objective: To investigate the morphology and immunohistochemical (IHC) expression of pseudostratified ependymal tubules in ovarian mature teratoma (MT). Methods: Five cases of ovarian MT with pseudostratified ependymal tubules were collected from Shenzhen Hospital(Futian) of Guangzhou University of Chinese Medicine and the Eighth Affiliated Hospital of Sun Yat-sen University from March 2019 to March 2022. In addition, 15 cases of ovarian MT with monolayer ependymal epithelium from Shenzhen Hospital (Futian) of Guangzhou University of Chinese medicine and seven cases of immature teratoma (IMT) from Hainan Provincial People's Hospital from March 2019 to March 2022 were collected as control. The morphologic characteristics and immunophenotypes of pseudostratified ependymal tubules, monolayer ependymal epithelium, and primitive neural epithelial tubules were observed and compared by H&E stain and IHC expression pattern of genes related to the differentiation status of neuroepithelium, namely SALL4, Glypican3, nestin, SOX2, Foxj1, and Ki-67. Results: Mean age of the five patients of ovarian MT with pseudostratified ependymal tubules was 26 years (range from 19 to 31 years). Two tumors were located in the left ovary and three in the right. All five cases were excised, and clinical follow-up was available (mean follow-up 1.5 years; range 0.5 to 3 years). No recurrence was noted in any cases. The pseudostratified ependymal tubules of ovarian MT, which were lined with columnar or oval epithelia up to 4-6 layers, were morphologically similar to the primitive neuroepithelial tubules of IMT and different from monolayer ependymal epithelium of ovarian MT. By immunohistochemistry, SALL4 and Glypican3 were negative, Foxj1 was positive and Ki-67 index was lower in the pseudostratified ependymal tubules and the monolayer ependymal epithelium of ovarian MT. However, the primitive neuroepithelial tubules of IMT showed variably expression of SALL4 and Glypican3, were negative for Foxj1 and high Ki-67 index. All the above three groups expressed nestin and SOX2. Conclusions: The pseudostratified ependymal tubules of ovarian MT, which have morphological similarities to the primitive neuroepithelial tubules of IMT, are similar to the monolayer ependymal epithelia of the MT in immunophenotype. IHC assessment of Foxj1 and Ki-67 is helpful to differentiate the pseudostratified ependymal tubules of ovarian MT from the primitive neuroepithelial tubules of IMT.
Female ; Humans ; Young Adult ; Adult ; Nestin ; Ki-67 Antigen ; Immunohistochemistry ; Ovarian Neoplasms/pathology* ; Teratoma/pathology*

OBJECTIVE: Human embryonic stem (ES) cells have a great potential in regenerative medicine and tissue engineering. The human ES cells could be differentiated into specific cell types by treatments of growth factors and alterations of gene expressions. However, the efficacy of guided differentiation and isolation of specific cells are still low. In this study, we characterized isolated cells from differentiated human ES cells by magnetic activated cell sorting (MACS) system using specific antibodies to cell surface markers. METHODS: The undifferentiated hES cells (Miz-hESC4) were sub-cultured by mechanical isolation of colonies and embryoid bodies were spontaneously differentiated with DMEM containing 10% FBS for 2 weeks. The differentiated cells were isolated to positive and negative cells with MACS system using CD34, human epithelial antigen (HEA) and human fibroblast (HFB) antibodies, respectively. Observation of morphological changes and analysis of marker genes expression were performed during further culture of MACS isolated cells for 4 weeks. RESULTS: Morphology of the CD34 positive cells was firstly round, and then it was changed to small polygonal shape after further culture. The HEA positive cells showed large polygonal, and the HFB positive spindle shape. In RT-PCR analysis of marker genes, the CD34 and HFB positive cells expressed endodermal and mesodermal genes, and HEA positive cells expressed ectodermal genes such as NESTIN and NF68KD. The marker genes expression pattern of CD34 positive cells changed during the extension of culture time. CONCLUSION: Our results showed the possibility of successful isolation of specific cells by MACS system from undirected differentiated human ES cells. Thus, MACS system and marker antibodies for specific cell types might be useful for guided differentiation and isolation of specific cells from human ES cells.
Antibodies ; Ectoderm ; Embryoid Bodies ; Endoderm ; Fibroblasts ; Gene Expression ; Humans* ; Intercellular Signaling Peptides and Proteins ; Mesoderm ; Nestin ; Regenerative Medicine ; Tissue Engineering