This study was aimed to investigate the biological characteristics of osteoblasts and their hematopoietic supportive function by using human fetal osteoblastic cell line 1.19 (hFOBs) as a model. The pluripotency markers (Oct-4, Rex-1, hTERT) of hFOBs were analyzed by RT-PCR, the multilineage differentiation experiments were conducted in vitro. Flow cytometry (FCM) was used to identify the surface markers of hFOBs, and RT-PCR was used to analyze their hematopoietic cytokine expression in comparison with bone marrow mesenchymal stem cell (BM-MSC). The results showed that hFOBs expressed several ESC pluripotency markers including Oct-4 and Rex-1, except hTERT. Moreover, hFOBs could also undergo multilineage differentiation into the mesodermal lineages of adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Both hFOBs and BM-MSC expressed CD44, CD73 (SH3), CD105 (SH2) and CD90 (Thy1), and lack expression of CD34, CD45, or HLA-DR surface molecules. In addition, both hFOBs and BM-MSC expressed SCF, IL-6, and SDF-1alpha mRNA, but only hFOBs could express GM-CSF and G-CSF. It is concluded that human fetal osteoblastic cell line 1.19 may provide a good model to study the osteoblastic regulation role in hematopoiesis in vitro.
Cell Differentiation ; physiology ; Cell Line ; Fetus ; Hematopoiesis ; physiology ; Humans ; Mesenchymal Stromal Cells ; cytology ; physiology ; Models, Biological ; Osteoblasts ; cytology ; physiology

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

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

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 immortalize human articular chondrocytes (HACs) using gene transfection and to maintain stable phenotype of transformed HACs after induction.

METHODSHACs were transfected with the retroviral vector pLXSN encoding human papillomavirus 16E7 (HPV16E7), and the transformed clones were sorted and proliferated. Karyotype analysis, clone forming tests and nude mice tumor forming tests were applied to check the characteristics of the transformation. Type II collagen of transformed chondrocytes was inducted with free serum medium (FSM) supplemented with nutridoma-sp and ascorbate.

RESULTSImmortalized HACs were isolated with fifty passages achieved. The HPV16E7 transformed cells were confirmed to be benign. Induction of FSM with nutridoma-sp and ascorbate promoted type II collagen of transformed chondrocytes to the high levels of normal chondrocytes.

CONCLUSIONHACs transformed with HPV16E7 survive for long periods in vitro, and type II collagen can maintain stability after induction.

Cells, Cultured ; Chondrocytes ; physiology ; Collagen Type II ; analysis ; Fetus ; Humans ; Knee Joint ; cytology ; Phenotype ; Transfection

One of the differences between fetal and adult skin healing is the ability of fetal wounds heal without contraction and scar formation. Extracellular matrix (ECM) provides a substratum for cells adhesion, migration, and proliferation and can directly influence the form and function of cells. As motility is essential for many important biological events, including wound healing, inflammatory response, embryonic development, and tumor metastasis, this study was designed to compare the motilities cultured dermal fetal and neonatal fibroblasts in the extracellular matrix. The motility of cultured fetal and neonatal fibroblasts was compared using a video-microscopy system that was developed in combination with a self-designed CO2 mini-incubator. To determine migration speed, cells were viewed with a 4X phase-contrast lens and video recorded. Images were captured using a color CCD camera and saved in 8-bit full-color mode. We found that cultured fetal fibroblasts move faster than neonatal fibroblast on type I collagen (fetal fibroblast, 15.1 micrometer/hr; neonatal fibroblast, 13.7 micrometer/hr), and in fibronectin (fetal fibroblast, 13.2 micrometer/hr; neonatal fibroblast, 13.0 micrometer/hr) and hyaluronic acid (fetal fibroblast, 11 micrometer/hr; neonatal fibroblast, 9.8 micrometer/hr).
Cell Movement ; Cells, Cultured ; Comparative Study ; Extracellular Matrix/*physiology ; Fetus/physiology ; Fibroblasts/*physiology ; Human ; Infant, Newborn ; Skin/cytology/*embryology ; *Skin Physiology

To characterize the background current in fetal human nasopharyngeal epithelial cells and clarify its relationship with volume activated Cl(-) currents (I(Cl,vol)), whole-cell patch clamp and cell imaging techniques were employed. Under isotonic conditions, a background current [(5.9+/-2.1) pA/pF at +80 mV, n=21] was detected. The current presented a weak outward rectification and a negligible time-dependent inactivation. The current-voltage relationship showed that the reversal potential of the background current [(-0.73+/-1.7) mV, n=21] was close to the calculated equilibrium potential for Cl(-)(-0.9 mV). Application of extracellular hypertonic stimulation (440 mOsmol/L) suppressed the current by (59.6+/-7.1)% and the inhibition was reversible after returned to isotonic conditions. Bathing the cells in hypotonic solution (160 mOsmol/L) induced a volume-sensitive Cl(-) current. The Cl(-) channel blockers, tamoxifen (20 micromol/L) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (100 micromol/L), inhibited the background current by (74.0+/-5.2)% (P<0.01, n=5) and (60.9+/-8.9)% (P<0.01, n=6) at +80 mV and increased basal cell volume by (107.7+/-2.9)% (P<0.01, n=25) and (104.4+/-2.4)% (P<0.01, n=19), respectively. The data indicate that Cl(-) current is an important component of the background current in fetal human nasopharyngeal epithelial cells. The background Cl(-) current is involved in volume activated Cl(-) current and basal cell volume regulation.
Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Electrophysiology ; Epithelial Cells ; cytology ; metabolism ; physiology ; Fetus ; Humans ; Nasopharynx ; cytology ; Nitrobenzoates ; pharmacology ; Patch-Clamp Techniques ; Tamoxifen ; pharmacology

OBJECTIVETo investigate the expression patterns of surfactant protein B (SP-B) and its role in the development of human fatal lung epithelial cells.

METHODSHuman fetal lung tissues were obtained from 37 fetuses of 10-34 weeks at abortion with parental consent and from two newborn infants who died of non-pulmonary causes. SP-B expression in the lung tissues was examined by immunohistochemistry.

RESULTSSP-B was detected in the cytoplasm of nonciliated columnar epithelial cells of the human fetal lung in as early as the 16th week of gestation. The positive reaction of SP-B was enhanced during canalicular stages and was more intense in the distal than in the proximal airway epithelium. From the 25th week to the prenatal stage, SP-B expression underwent no significant changes in the primitive alveolar stage, but increased remarkably after birth.

CONCLUSIONThe expression and secretion of SP-B reflects the maturation of the epithelial cells in human fatal lungs, and may closely associate with the survival ability of the newborn infants.

Cell Survival ; physiology ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Fetus ; Humans ; Infant, Newborn ; Lung ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Surfactant-Associated Protein B ; biosynthesis ; physiology

This study was aimed to investigate if human heart harbored a population of primitive undifferentiated cells with the characteristics of MPC. Cells were isolated from human fetal heart and were cultured under conditions appropriate for bone marrow-derived MPCs. Their morphology, phenotypes and functions were tested by methods developed for MPC from other sources. The results showed that morphologically, cells were spindle shaped and resembled fibroblasts. In their undifferentiated state, cells were CD73, CD105, CD29, CD44, HLA-ABC, CD166 positive and CD45, CD34, CD86, HLA-DR negative. When cultured in adipogenic, osteogenic or chondrogenic media, cells differentiated into adipocytes, osteocytes and chondrocytes respectively. They could be extensively expanded in vitro and exhibited very low immunogenicity as evaluated by T cell proliferation assays. It is concluded that cells isolated from fetal heart possess similarity to their adult and fetal bone marrow counterparts in morphologic, immunophenotypic, and functional characteristics.
Bone Marrow Cells ; cytology ; Cell Adhesion ; Cell Differentiation ; Cells, Cultured ; Fetal Heart ; cytology ; Fetus ; Humans ; Mesenchymal Stromal Cells ; cytology ; immunology ; Multipotent Stem Cells ; physiology

OBJECTIVETo study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.

METHODSHuman chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.

RESULTSAll these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05).

CONCLUSIONSComparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.

Animals ; Cartilage ; cytology ; metabolism ; physiology ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Fetus ; cytology ; Humans ; Mice, Nude ; Proteome ; metabolism ; Proteomics ; Tissue Engineering ; methods ; Tissue Scaffolds