Ex vivo expansion of functional human UCB-HSCs/HPCs by coculture with AFT024-hkirre cells.

Kiaa1867 (human Kirre, hKirre) has a critical role in brain development and/or maintenance of the glomerular slit diaphragm in kidneys. Murine homolog of this gene, mKirre expressed in OP9 and AFT024 cells could support hematopoietic stem cells/hematopoietic progenitor cells (HSC/HPC) expansion in vitro. HKirre is also expressed in human FBMOB-hTERT cell line and fetal liver fibroblast-like cells but its function has remained unclear. In this paper, we cloned a hKirre gene from human fetal liver fibroblast-like cells and established a stably overexpressing hKirre-AFT024 cell line. Resultant cells could promote self-renewal and ex vivo expansion of HSCs/HPCs significantly higher than AFT024-control cells transformed with mock plasmid. The Expanded human umbilical cord blood (hUCB) CD34(+) cells retained the capacity of multipotent differentiation as long as 8 weeks and successfully repopulated the bone marrow of sublethally irradiated NOD/SCID mice, which demonstrated the expansion of long-term primitive transplantable HSCs/HPCs. Importantly, hkirre could upregulate the expressions of Wnt-5A, BMP4, and SDF-1 and downregulate TGF- ß with other hematopoietic growth factors. By SDS-PAGE and Western Blot analysis, a ~89 kDa protein in total lysate of AFT024-hKirre was identified. Supernatants from AFT024-hkirre could also support CD34(+)CD38(-) cells expansion. These results demonstrated that the AFT024-hKirre cells have the ability to efficiently expand HSCs/HPCs.

Microgravity inhibits resting T cell immunity in an exposure time-dependent manner.

BACKGROUND: Decline immune function is well documented after spaceflights. Microgravity is one of the key factors directly suppressing the function of immune system. Though T cell immune response was inhibited by microgravity, it is not clearly whether activation would be inhibited after a pre-exposure of microgravity on T lymphocytes at the resting state. METHODS: We herein investigated the response ability of resting CD4⁺ and CD8⁺ T cells experiencing pre-exposure of modeled microgravity (MMg) for 0, 8, 16 and 24 hrs to concanavalin A (ConA) stimulation. The phenotypes and subsets of immune cells were determined by flow cytometry. RESULTS: Both CD4⁺ and CD8⁺ T cells with an MMg pre-exposure exhibited decreased expressions of activation-markers including CD25, CD69 and CD71, inflammatory cytokine secretion and cell proliferation in response to ConA compared with T cells with 1g controls in an MMg exposure time- dependent manner. Moreover, short term MMg treatment caused more severe decreased proliferation in CD4⁺ T cells than in CD8⁺ T cells. CONCLUSIONS: MMg can directly impact on resting T cell subsets. CD4⁺ T cells were more sensitive to the microgravity inhibition than CD8⁺ T cells in respect of cell proliferation. These results offered new insights for the MMg-caused T cell functional defects.

Microgravity inhibition of lipopolysaccharide-induced tumor necrosis factor-α expression in macrophage cells.

OBJECTIVE AND DESIGN: Microgravity environments in space can cause major abnormalities in human physiology, including decreased immunity. The underlying mechanisms of microgravity-induced inflammatory defects in macrophages are unclear. MATERIAL OR SUBJECTS: RAW264.7 cells and primary mouse macrophages were used in the present study. Lipopolysaccharide (LPS)-induced cytokine expression in mouse macrophages was detected under either simulated microgravity or 1g control. METHODS: Freshly isolated primary mouse macrophages and RAW264.7 cells were cultured in a standard simulated microgravity situation using a rotary cell culture system (RCCS-1) and 1g control conditions. The cytokine expression was determined by real-time PCR and ELISA assays. Western blots were used to investigate the related intracellular signals. RESULTS: LPS-induced tumor necrosis factor-α (TNF-α) expression, but not interleukin-1ß expression, in mouse macrophages was significantly suppressed under simulated microgravity. The molecular mechanism studies showed that LPS-induced intracellular signal transduction including phosphorylation of IKK and JNK and nuclear translocation of NF-κB in macrophages was identical under normal gravity and simulated microgravity. Furthermore, TNF-α mRNA stability did not decrease under simulated microgravity. Finally, we found that heat shock factor-1 (HSF1), a known repressor of TNF-α promoter, was markedly activated under simulated microgravity. CONCLUSIONS: Short-term treatment with microgravity caused significantly decreased TNF-α production. Microgravity-activated HSF1 may contribute to the decreased TNF-α expression in macrophages directly caused by microgravity, while the LPS-induced NF-κB pathway is resistant to microgravity.

MiR-17 partly promotes hematopoietic cell expansion through augmenting HIF-1α in osteoblasts.

BACKGROUND: Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment, of which osteogenic cells play a crucial role. While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34(+) cell expansion and maintain their multipotency. It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34(+) cells compared to other niche associated factors. More interestingly, the specific erythroid lineage expansion of CB CD34(+) cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down. CONCLUSION/SIGNIFICANCE: Our data demonstrated that CB CD34(+) cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts.

Labeling of hematopoietic stem cells by Tat peptide conjugated quantum dots for cell tracking in mouse body.

Fluorescent quantum dots have great potential to act as labeling tools in biological research, especially cellular tracking and imaging. Tat peptide conjugated quantum dots were introduced into living human hematopoietic stem cells (HSCs). The internalized quantum dots were assessed by laser confocal microscope and flow cytometer. The quantum dots labeled HSCs were injected intravenously into the tail veins of NOD/SCID beta2M null mice. The tissue collections in the major organs were examined with fluorescence microscope to assess the distribution of transplanted stem cells. HSCs with internalized quantum dots offer a promising approach for stem cell transplantation, which will hold a significant impact on stem cell based therapy for several disorders, especially to cure leukemia in current China.

Applications of fluorescent quantum dots to stem cell tracing in vivo.

Fluorescent quantum dots have great potential to act as labels in biological research, especially cellular tracking in vivo. Here, small thiol molecules-capped CdSe/ZnS quantum dots attached to Tat peptide were used for label agents, and introduced into living stem cells. The stem cells labeled with quantum dots were intravenously injected into the tail veins of NOD/SCID beta2M null mice, and whose tissue sections of major organs of null mice were examined with fluorescence microscope to assess the distribution of transplanted stem cells. Stem cells with internalized quantum dots offer a promising approach in stem cell transplantation, which will hold a significant impact on stem cell based therapy for several disorders.

[Laser tweezers Raman spectroscopy analysis of liver cancer tissue].

Single cell laser tweezers Raman spectroscopy (LTRS) has been applied to biology field. In the present article, the authors measured the spectra of liver cancer cells, para-cancer cells and normal hepatocytes using single cell laser tweezer Raman spectroscopy (LTRS) system and compared their average spectra changes. The results showed that the laser tweezers Raman spectroscopy could differentiate specimens of different pathological changes from liver tissue studied. The 1 070 and 1 266 cm(-1) peaks obtained from normal hepatocytes were more visible than the same two peaks obtained from liver cancer and para-cancer specimen. The 1 445 cm(-1) peak of normal hepatocytes was higher than that of liver cancer cells and para-cancer cells. It is known that the 1 070 cm(-1) peak represents lipids and nucleic acids, while 1 266 and 1 445 cm(-1) peaks represent lipids and proteins. So, these peak changes may directly reflect the changed biomaterials related to liver carcinogenesis. Thus, single cell laser tweezer Raman spectroscopy may be a nondestructive, rapid and good method to measure and analyze different pathological specimens from liver cancer.

Human leukocyte antigen-G1 inhibits natural killer cytotoxicity through blocking the activating signal transduction pathway and formation of activating immunologic synapse.

Nonclassical major histocompatibility complex (MHC) class I molecule human leukocyte antigen (HLA)-G is normally expressed on the placental cells, especially fetal endothelial cells and invasive cytotrophoblast cells at the maternal-fetal interface. This antigen meditates immune tolerance in pregnancy through interaction with immune cells including natural killer (NK) cells. In this study, we investigated the mechanisms underlying HLA-G1-mediated inhibition of NK cytotoxicity using HLA-G1-transfected K562 cells and NK92 cells. We found that inhibition of NK cytotoxicity by HLA-G1 was associated with decreased formation of NK-target cell conjugates and defective formation of immunologic synapse, as characterized by actin depolarization and perforin immobilization in nonactive NK cells. HLA-G1 engagement induced dephosphorylation of Vav by tyrosine phosphatase-1 (SHP-1), and thus blocked the Syk-->MEK/ERK activating signaling pathway in activating NK cells. These results indicate that HLA-G1 inhibits NK cytotoxicity by blocking activating signal transduction pathway, which is required for the formation of activating immunologic synapse.

Gastric cancer cells in collagen gel matrix: three-dimensional growth and differential expression of adhesion molecules (CD44s, CD54, E-cadherin).

To characterize the growth of human gastric cancer cells in collagen gel matrix and adhesive status of the cells in comparison with conventional monolayer cells. Three kinds of human gastric cancer cell lines (BGC823, SGC7901, and MKN28) were cultured alone or co-cultured with normal human fibroblasts in collagen gel matrix, and their cell cycle, metabolic function, and the expression of adhesive molecules (CD44s, CD54, and E-cadherin) were analyzed by flow cytometry or other methods. Two of three cell lines (BGC823 and SGC7901) and their co-cultures showed multilayer growth in collagen gel matrix, and their growth and metabolism rate became slow and the cell adhesion molecules (CAMs) expression was down regulated. Gastric cancer cell alone or with fibroblasts in collagen gel matrix showed distinct growth feature when compared with monolayer cells, which represent two kinds of different experimental models. BGC823 and SGC7901 cells growing in three-dimension may recur some characteristics of their original solid tumor in vivo with the invasive or metastatic ability. According to different aims, it should pay great care in choice of experimental model to get more reasonable results.

Applications of mesenchymal stem cells labeled with Tat peptide conjugated quantum dots to cell tracking in mouse body.

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells. The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy, laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots could enter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into the tail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots was observed primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain, the heart, or the kidney.

Establishment and characterization of a human telomerase catalytic subunit-transduced fetal bone marrow-derived osteoblastic cell line.

The fate of human hematopoietic stem cells (HSCs)/progenitor cells (HPCs) is influenced by bone marrow (BM) stromal cells. To investigate the role of stromal cells in the hematopoietic support, we have transduced human fetal BM stromal cells (FBMSCs) with a human telomerase catalytic subunit (hTERT). One of the resultant cell lines was identified as osteoblasts, because it contained mineral deposits and constitutively expressed osteogenic genes osteocalcin, osteopontin, collagen type I, osteoblast marker alkaline phosphatase, but not marrow stromal cell marker STRO-1 and CD105. The hTERT-transduced fetal BM-derived osteoblastic cells (FBMOB-hTERT) can actively maintain the capacity of self-renewal and multipotency of HSCs/HPCs at least partly through transcriptional up-regulation of hematopoietic growth factors such as stem cell growth factors (SCFs) and Wnt-5A during interaction with HSCs/HPCs. The enhanced transcription of SCFs and Wnt-5A appears to be mediated by CD29 signaling. Moreover, the FBMOB-hTERT cells seem superior to primary FBMSCs in supporting hematopoiesis, because they are more potent than primary FBMSCs in supporting the ex vivo expansion and long-term culture initiating cells activity of HSCs. The FBMOB-hTERT cell line has been maintained in vitro more than 125 population doublings without tumorigenicity. The results indicate that the FBMOB-hTERT is useful for the study of molecular mechanisms by which osteoblasts support hematopoiesis.

NIR Raman spectroscopic investigation of single mitochondria trapped by optical tweezers.

Raman spectroscopy is a vibration spectroscopic technique that has been widely used to probe biochemical changes of biological sample such as tumor tissue, blood cells, bacteria and yeast. Here, we applied near-infrared Raman spectroscopy to analyze the chemical composition changes of intact or swollen mitochondria induced by calcium ions. We used a confocal Laser Tweezers Raman Spectroscopy (LTRS) system that combined optical trapping and near infrared Raman spectroscopy to confine a single mitochondrion and consequently measure its Raman spectra following the addition of calcium ion solution. We analyzed Raman spectra of mitochondria isolated from rat liver, heart muscle and kidney, respectively. The major Raman peaks at 1654, 1602, 1446, 1301 and 1226 cm(-1) were observed from individual intact mitochondria. We examined the differences in near infrared spectra between intact and Ca(2+) damaged mitochondria. We found that after the exposure of the intact mitochondria to the 100 muM Ca(2+) solution the band of 1602 cm(-1) decreased very rapidly in the first period and then disappeared after 30minutes, while the intensities of the phospholipids and protein bands changed slowly in the first period and then suddenly disappeared, corresponding to the Ca(2+) induced swelling process. These results demonstrate the potential of LTRS technique as a valuable tool for the study of bioactivity and molecular composition of mitochondria.

[Three-dimensional spheroid model for cultivating WB-F344 cells in simulated microgravity].

Three-dimensional (3D) culture of cells could closely mimic the in vivo situation with regard to cell function and microenvironment compared with plane monolayer cultured cells. In this paper, we established 3D culture of rat WB-F344 cells with rotary cell culture system (RCCS) to simulate microgravity environment, and examined cells proliferation, morphology, microstructure, E-cadherin protein quantity and mRNA expression of adhesion molecules by count the number of cells, optical microscope, transmission electron microscope and reverse transcriptase-polymerase chain reaction (RT-PCR). The results demonstrated that cells were polyhedron with lots of micovilli and mitochondria, which grow well and packed together densely to form irregular aggregates. Adjacent cells were connected with desmosome and tight junction. With the regard, the aggregates behaved 3D growth characteristics. Moreover, compared with control, mRNA level of Fibronectin and E-cadherin protein were increased, the changes maybe is the part mechanism in this microgravity simulated cells culture models which strengthened cells junction. This rotating 3D model might facilitate the study of interactions of cell-cell, cell-matrix and the mechanisms.

Differential expression of HNF4alpha isoforms in liver stem cells and hepatocytes.

The hepatocyte nuclear factor (HNF4alpha) has been implicated in liver development and hepatocellular differentiation. The HNF4alpha expression in liver stem cells has not yet been fully investigated. Here, we describe the expression characterization of HNF4alpha in liver stem cells isolated from 2-acetylaminofluorene/partial hepatectomy (AAF/PH) rat model by a combination of density-gradient centrifugation and selective enzymatic digestion. The obtained cells were identified by flow cytometry (FCM) immediately and then by immunocytochemisty and reverse transcription-polymerase chain reaction (RT-PCR) after 5 d culture. The cultured stem cells were subjected to analysis of the expression of various HNF4alpha isoforms using RT-PCR, Western blotting, and electrophoretic mobility shift assay (EMSA). Results showed that HNF4alpha isoforms, alpha1, alpha2, alpha7, and alpha8 were present in liver stem cells, which contrasted with only alpha1 and alpha2 expression in hepatocytes. The distinct expression patterns of HNF4alpha isoforms in liver stem cells may contribute to maintain the "stemness" of these cells.

Rapid production of human KIR2DL4 extracellular domain and verification of its interaction with HLA-G.

Killer cell immunoglobulin-like receptor (KIR) 2DL4 is the only KIR member reported to be expressed by all human natural killer (NK) cells. It differs from other KIR members in both structure and function. Its specific interaction with HLA-G, a non-classical MHC class I molecule, has been suggested to play an important role in regulating NK cell-mediated cytotoxicity. However, this interaction is still in doubt. In addition, the soluble KIR2DL4 extracellular domain used in many studies was produced by eukaryotic expression, which is less efficient than prokaryotic expression. In this study, we describe a method of rapid production a large amount of soluble KIR2DL4 extracellular domain based on a prokaryotic expression system. With this soluble KIR2DL4, we verified the interaction between KIR2DL4 and HLA-G1.

Improved performance of primary rat hepatocytes on blended natural polymers.

Alginate (Alg), chitosan (Chi), collagen (Col), gelatin (Gel), and the mixtures of every two of them were screened to determine their suitability for hepatocyte culture. The test materials were fabricated as films and then evaluated on the basis of their abilities to promote the attachment and functions of rat hepatocytes cultured on them. Cellular attachment on Col and Gel was favorable. However, cellular viability, cytoskeleton organization and function, as evaluated by albumin production, ureagenesis, and enzyme activity of cytochrome P450 as well as expression levels of hepatocyte nuclear factor 4 alpha deteriorated. Reverse cellular behavior was observed on Alg and Chi. Two blends, composed of Chi and Col or Gel, were found to be superior to other materials and sustained viability and differentiated functions of hepatocyte.

The existence of epithelial-to-mesenchymal cells with the ability to support hematopoiesis in human fetal liver.

The fetal liver is the major hematopoietic organ during mid-gestation, and it is also a source of stem cells that exist in a complex environment. In this study, we isolated a population of actively replicating cells with the characteristic of the epithelial-to-mesenchymal transition (EMT) from fetal liver. These cells were identified with the epithelial markers, including alpha-fetoprotein (AFP), albumin (ALB), cytokeratins (CK) 7, and CK18, as well as the mesenchymal markers, such as alpha-smooth muscle actin (ASMA), CD29, CD44, CD49, CD54, collagen I and osteopontin (OPN). Furthermore, they also expressed some hematopoiesis-related genes. In addition, the cell population had the ability to retain hematopoietic stem cells (HSCs) in an undifferentiated state in vitro during cytokine-stimulated proliferation. These results provide an insight about early human liver development and may also help to understand hematopoiesis in the fetal liver.

Chitosan/gelatin composite microcarrier for hepatocyte culture.

Solid and porous chitosan/gelatin (CG) composite microcarriers were prepared by a water-in-oil emulsion process with additional freezing and lyophilization. Adult rat hepatocytes (10(6) cells ml(-1)) attached on CG microcarriers maintained at least 15 d of viability and differentiated functions. Over 15 d, unimmobilized hepatocytes released 1.34-fold less lactate dehydrogenase (LDH), and retained 1.63-, 1.51- and 1.28-fold higher albumin secretion, urea synthesis and 7-ethoxycoumarin deethylation activities, respectively, than those on collagen-coated microcarriers. The CG matrix is therefore a promising microcarrier for hepatocyte culture.

[Major role of stromal cell derived factor and its receptor CXCR4 in the mobilization and homing of hematopoietic stem/progenitor cells - review].

Stromal cell derived factor (SDF), expressing on bone marrow stromal cells is a CXC-type chemokine, which specifically chemoattracts hematopoietic stem cells (HSCs) expressing CXCR4. SDF plays important roles in homing and mobilizing of HSCs. In this paper the regulatory mechanism of SDF/CXCR4 in the HSC migration process is mainly reviewed.