OBJECTIVETo explore the preliminary methods of in vitro isolation, culture and identification of sebocytes and eccrine sweat gland cells from human fetal skin.

METHODSHuman fetal skin was digested with dispase or type II collagenase, and then by micro - sieving to isolate human sebaceous gland and eccrine sweat gland cells. DMEM/F12 (1: 1) was used as the basic culture medium, supplemented with fetal bovine serum, recombinant human epidermal growth factor, L-glutamine, Hydrocortisone, choleratoxin, penicillin and streptomycin as the medium for sebocytes, or fetal bovine serum, recombinant human epidermal growth factor, triiodothyronine, hydrocortisone, insulin, transferrin, sodium selenite to the medium for eccrine sweat gland duct cells. Primary cultures and subcultures were incubated at 37 degrees C in humidified atmosphere of 5% CO2/95% oxygen. Cell morphology was observed by inverted phase contrast microscopy, and the cultured cells were identified with cell clone efficiency determination. The cultured sebocytes were identified with oil red staining and CK4.62, Epithelia Membrane Antigen (EMA) immunohistochemistry staining. The cultured eccrine sweat gland duct cells were identified with CK7, CK19 immunohistochemistry staining.

RESULTSThe isolated sebocytes and eccrine sweat gland cells from human fetal skin could grow by adhering to the wall and proliferate in vitro. The cell clone efficiency of human fetal sebocytes was 2.7%, which was obviously lower than that of human fetal keratinocytes (8.0%, P < 0.01). There was no obvious difference in the cell clone efficiency between human fetal eccrine sweat gland cells (7.3%) and human fetal keratinocytes (7.7%, P > 0.05) . The results of oil red staining indicated that a small quantity of lipid droplets in sebocytes, and immunohistochemistry staining of CK4.62, EMA were positive in subculture sebocytes. The immunohistochemistry staining of CK7, CK19 was positive in subculture eccrine sweat gland duct cells.

CONCLUSIONIn vitro cultured human fetal sebocytes and eccrine sweat gland duct cells displayed the markers and biological characteristics of epithelial lineage, but human fetal sebocytes proliferated more

Cell Culture Techniques ; Eccrine Glands ; cytology ; Fetus ; cytology ; Humans ; Sebaceous Glands ; cytology ; Skin ; cytology ; Vernix Caseosa ; cytology

Animals ; Cell Differentiation ; Fetus ; cytology ; Hematopoietic Stem Cells ; cytology ; Hepatocytes ; cytology ; Humans ; Liver ; cytology ; Stem Cells ; cytology ; physiology

Active migration of immature neurons occurs when fragments of human foetal cerebral tissues are explanted as organotypic cultures. The sequence of events during neuronal migration is orderly and consistent under different cultural conditions as evidenced by continuous time-lapse cinematographic studies. Migrating neurons utilize astrocytes to anchor neurites, and move in clusters on or along the processes of astrocytes or other neurons. Translocation of neuronal soma is accomplished by nuclear movement within extended neurites. A unique junction develops between neurites and astrocytic membrane during early phases in culture to suggest a special affinity of neurons to astrocytes. It is concluded from these observations that immature neurons have inherent capacity for active migration in-vitro; preferentially utilize astrocytes and astrocytic processes for anchoring as well as for directional guidance during migration; and translocate their soma by nuclear movement within extended neurites. It is suggested that similar mechanisms may be at play during migration of postmitotic neurons in developing cerebral cortex in human.
Astrocytes/cytology ; Brain/cytology ; Brain/embryology* ; Cell Movement ; Fetus ; Human ; Neural Conduction ; Neurons/cytology* ; Tissue Culture

OBJECTIVETo investigate the phenotypic characteristics of human fetal liver cells (FLCs) and to obtain the homogenous hepatic progenitors with cloning.

METHODSImmunofluorescence and flow cytometry were used to determine the phenotypes of the FLCs. The proliferating colonies were isolated using clone ring in different culture conditions. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the mRNA expression after further cultivation.

RESULTSThe cultured FLCs showed a non-typical epithelial morphology. The positive rate for hepatic cell specific markers alpha-fetoprotein (AFP), albumin (Alb), cytokeratin 8 (CK8) and CK19 were approximately 28.1%, 84.7%, 55.1% and 9.1% respectively. Furthermore, the FLCs expressed the hematopoietic stem cell markers CD34 and CD45 with percentages of 0.04% and 0.09%. 71.8% and 75.3% of the FLCs were positive for the mesenchymal cell marker CD105 and CD166. Most of the colonies showed an elongated morphology, some with an unregular spreading-out morphology, only a small number of colonies with an epithelial-like morphology. RT-PCR results showed that among the 19 colonies obtained after further cultivation and the percentages of epithelial cell adhesion molecule (EpCAM), AFP, Alb and CK19 were 52.6%, 21.1%, 52.6% and 84.2%, respectively.

CONCLUSIONSThe clonal culture system is beneficial to obtain the homogenous hepatic progenitor cells from the heterogeneous culture of FLCs.

Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Fetus ; cytology ; Hepatocytes ; cytology ; Humans ; Stem Cells ; cytology

Male germ stem cells (mGSCs), which is in testis after sex differentiation, derive from primordial germ cells. In this study, bovine mGSCs were isolated from testis of 20 weeks fetuses. Number of CD9 positive cells of the cells through two-steps adhering plates velocity different was 95.8% by flow cytometer. The carina-type cells clones and the plane-type cells clones appeared in co-cultured system. One cells lines had been successively maintained for 4 passages, and the cells clusters showed AKP positive staining. The cells clusters showed nest-shape in third passage showed SSEA1 and Oct-4 positive staining. These cells can also spontaneously differentiate into c-kit positive staining germ cells, and the cells were directional induced to formaactin positive staining cardiac-like cells cluster and NF positive staining neuron-like cells. The conclusion showed that male germ stem cells from 20 weeks bovine fetuses could be in vitro formed like embryonic stem cells.
Animals ; Cattle ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Fetus ; cytology ; Male ; Pluripotent Stem Cells ; cytology ; Spermatozoa ; cytology

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSC-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expressions of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens, such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and were also tested by RT-PCR at the mRNA level. Then, hfBM-MSCs were induced to differentiate toward neuron cells, adipose cells, and islet B cells under certain conditions. It was found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G(0)/G(1) phase respectively. hfBM-MSCs expressed CD44, CD106 and adhesion molecule CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose-like cells, and islet B-like cells and they were tested to be positive for related characteristic markers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.
Bone Marrow Cells ; cytology ; Cell Proliferation ; Embryonic Stem Cells ; cytology ; Fetus ; Germ Layers ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo induce nonparenchymal mesenchymal stem cells (NPMSCs) differentiating into functional hepatocyte-like cells in vitro, and to identify the molecular biology and functional characteristics of those hepatocyte-like cells.

METHODSHuman NPMSCs were isolated and cultured with cell culture technique. NPMSCs were induced (on 1% Matrigel as a matrix and then submitted to 2.5 mmol/L AZA pretreatment for 10-12 h), by adding HGF 10 microg/L + FGF4 10microg/L + HGM into the culture medium. The characteristics of proliferation and growth of human NPMSCs were studied with methyl thiazolyl tetrazolium (MTT). The phenotypes of NPMSCs were identified by flow cytometry, immunohistochemistry and RT-PCR. Albumin (Alb) levels in culture supernatants were determined with ELISA. Staining for glycogen of undifferentiated NPMSCs and NPMSCs derivated hepatocyte-like cells was conducted with periodic acid-Schiff (PAS) test.

RESULTSGrowth and division of adherent cells obtained from fetal livers were good and the amount of NPMSCs resourced from each fetus could be amplified to 109 cells after 10 serial subcultivations. The phenotype of NPMSCs was CD166 positive and CD34 negative. The shape of NPMSCs plated on Matrigel with FGF4 and HGF changed from long fusiform to polygonal or round on days 21-28. The rate of cell rounding was 40% and the ratio of dikaryocytes was 5%. Immunohistochemical and RT-PCR detection showed that undifferentiated NPMSCs expressed few alpha fetoprotein (AFP) and AFP mRNA, and did not express any of the liver-specific transcription factors or cytoplasmic markers. Many cells in early induction expressed GATA4, AFP and CK18 proteins and their mRNAs, and their expressions were reduced at the late induction, but the expressions of Alb, CK18, GST-and hepatocyte transcription factor HNF1increased gradually. The ratio of Alb and CK18 positive cells was 60%. Undifferentiated NPMSCs did not produce Alb. Alb production by induced NPMSCs increased in a time-dependent manner. Glycogen storage was first seen on day 14, and maximum levels were seen after day 28.

CONCLUSIONSThere are MSCs among nonparenchymal cells of fetal livers. A high ratio of hepatocyte-like cells was obtained under our induction condition. NPMSCs differentiate firstly into hepatocyte precursors, and then differentiate into mature hepatocytes and hepatocyte-like cells with positive hepatocyte markers. The induced NPMSCs have hepatocyte specific functional features.

Cell Differentiation ; Cell Separation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Fetus ; cytology ; Hepatocytes ; cytology ; Humans ; Liver ; embryology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo isolate and culture mesenchymal stem cells (MSCs) from human fetal livers and describe their biological characteristics.

METHODSMSCs were acquired using an optimized method. Cell cycles and the immunophenotype of the cells were analyzed by flow cytometry. The osteogenic and adipogenic differentiations were induced and identified by specific stainings, and hepatic differentiation by morphology and RT-PCR.

RESULTSThe target cells derived from human fetal livers adhered to the plate with fibroblast-like morphology, whose surface markers were CD90, CD44, CD147 positive, and CD34, CD45, HLA-DR negtive. In the differentiation study, these cells could be induced to differentiate into osteogenic, adipogenic and hepatocyte-like cells.

CONCLUSIONMultipotent MSCs can be isolated and cultured from human fetal livers.

Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Fetus ; Humans ; Liver ; cytology ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo investigate the growth and development of nerve growth factor (NGF)-positive neurons in the cerebellum of midanaphase human fetus.

METHODSThe expression of the NGF-positive neurons in the cerebrum of human fetus was observed by immunohistochemical methods, and the integral absorbance (IA) was detected.

RESULTSBy the 3rd to 4th month of gestation, neurons was seen in the ependymal, central, and marginal plate of cerebellum; the nucleus was oval and the neurons had short and small processes. By the 5th to 7th month of gestation, the number of NGF-positive neurons increased, the expressions enhanced, the nucleus was round-, oval-, or fusiform-shaped, the neurons grew larger in size, and the Purkinje cells showed NGF-positive expression. By the 8th to 10th month of gestation, the NGF-positive expression was enhanced with deeper dying, the body of Purkinje cells grew larger gradually, and the number of NGF-positive neurons in the granular cell layer and molecular layer increased. IA of the cerebellar cortical neurons of the 3rd, 4th, 5th, 6th, 7th, and 8th month of gestation showed an increasing trend, and significant difference was observed (P < 0. 05).

CONCLUSIONNGF-positive neurons in the cerebellum play an important role for differentiation, proliferation, migration, and growth of neurons in the cerebellum.

Cerebellum ; cytology ; metabolism ; Fetus ; cytology ; metabolism ; Humans ; Nerve Growth Factor ; metabolism ; Neurons ; cytology ; metabolism ; Purkinje Cells ; metabolism

OBJECTIVETo investigate the expression of metabotropic glutamate receptor 5 (mGluR5) and its cellular distribution in the frontal cortex, ventricular zone (VZ) and subventricular zone (SVZ) in human fetuses.

METHODSAccording to the gestational age, the collected fetuses were divided into 4 groups, namely 9-11 weeks, 14-16 weeks, 22-24 weeks and 32-36 weeks. Brain tissue blocks including the frontal lobe or VZ/SVZ were prepared into slices, and the expression pattern and cellular distribution of mGluR5 in the frontal cortex and VZ/SVZ were observed by immunohistochemistry or immunofluorescence.

RESULTSmGluR5 immunoreactivity was present in the cell membrane in the frontal cortex, VZ and SVZ from the 9th to 36th weeks and the immunoreactivity in the marginal zone (MZ) and cortical plate (CP) was markedly stronger than that in VZ and SVZ. The cells expressing mGluR5 included neural stem/progenitor cells in the VZ and SVZ, immature neurons in the VZ and MZ, and numerous mature neurons in the CP.

CONCLUSIONmGluR5 is expressed by a variety of cells such as neural stem cells in the frontal cortex, VZ and SVZ in human fetus, suggesting a role of mGluR5 in the development of human cerebral cortex.

Cerebral Cortex ; cytology ; Cerebral Ventricles ; cytology ; metabolism ; Fetus ; cytology ; metabolism ; Frontal Lobe ; cytology ; metabolism ; Humans ; Neural Stem Cells ; cytology ; metabolism ; Receptor, Metabotropic Glutamate 5 ; Receptors, Metabotropic Glutamate ; metabolism