Expression Of SCF Research Articles

A New Immunodeficient Mouse Strain, NOD/SCID IL2Rγ−/− SGM3, Promotes Enhanced Human Hematopoietic Cell Xenografts with a Robust T Cell Component.

Abstract 3524Poster Board III-461Several strains of immunodeficient mice have been employed in the effort to model both normal and aberrant growth and differentiation of human hematopoietic cells in vivo. Despite numerous successes in this field, room remains for improvement. For example, robust models of in vivo differentiation and expansion of human erythroid and T cells are lacking or challenging at best. With the goal to further improve in vivo human hematopoietic cell study, we have crossed the NOD/SCID IL2Rγ knockout mice (NSG) with the NOD/SCID-SGM3 mice (NS-SGM; transgenic expression of human cytokines SCF, GM-CSF, and IL-3) to create a new strain of immunodeficient mice (NSG-SGM3). Uncultured umbilical cord blood CD34+ cells (UCB) were used to initiate xenografts in these three strains as well as NOD/SCID (NS) mice for comparison. Although xenografts with high human composition can be set up in NS mice, the graft, which is B-lymphoid biased without any measurable T cell component, tends to fade over time. In comparison, NSG mice offer longer sustained engraftment with a predominantly B-lymphoid bias and an inconsistent minor T cell component that is absent in NS mice. These mice also display increased levels of human cells in the periphery, allowing for easier measurement of the graft. Similar to NS grafts, NS-SGM3 grafts are initiated at high levels of chimerism, decrease significantly over time, lack T cells, and have a relatively low peripheral involvement. However, the graft consists of a more balanced myeloid/lymphoid distribution. The NSG-SGM3 combines some of the useful properties of the parent strains. Grafts remain at high, sustained levels through 16 weeks while maintaining a fairly balanced myeloid/lymphoid contribution. Chimerism in the NSG-SGM3 mice is readily detectable in the periphery as well as the BM. Remarkably, as early as 8 weeks post-transplant, we observed consistently high human T cell engraftment and expansion far superior to what we observed in NSG mice in parallel experiments using the same UCB samples. Flow cytometric analysis revealed the presence of numerous T cell sub-populations, suggesting significant recapitulation of human T cell development and function in NSG-SGM3 mice. Although it remains unclear as to why the unique combination of SGM3 cytokine expression and IL2Rγ knockout promotes enhanced human T cell engraftment and expansion, this mouse presents an improved opportunity to examine human T cell development and function in a xenograft setting as well as modeling T cell associated diseases including HIV infection, GVHD, and T cell leukemia. Disclosures:No relevant conflicts of interest to declare.

Tumour microvessel endothelial cell KIT and stem cell factor expression in human solid tumours

To assess KIT receptor tyrosine kinase and stem cell factor (SCF, KIT ligand) expression in tumour microvessel endothelial cells. KIT and SCF expression were studied in 248 human tumours consisting of 15 different histological types using immunohistochemistry and in situ hybridization for KIT messenger RNA. Moderate to strong intratumoral endothelial cell KIT expression was present in 11 of the 15 tumour types, and was most common in glioblastoma (58%), mixed embryonal carcinoma with teratoma (33%) and renal clear cell carcinoma (29%). Results of in situ hybridization were in line with those obtained with immunohistochemistry in the cases studied (n = 9). Marked SCF expression was uncommon in tumour endothelial cells, but frequent in perinecrotic tumour cells. Patients with glioblastoma with moderate to strong endothelial cell KIT expression had more favourable survival than those whose tumour showed little or no expression (P = 0.024). Glioblastoma patients whose tumour expressed SCF had an unfavourable outcome compared with those with tumour with weak or no expression (P = 0.034). Intratumoral microvessels of several types of human malignant tumours express KIT. Tumour cell SCF expression and absence of marked endothelial cell KIT expression are novel adverse prognostic features in glioblastoma.

Inverse correlation between Skp2 and p27Kip1 in normal endometrium and endometrial carcinoma

Cyclin-dependent-kinase (cdk) inhibitor, p27(Kip1) (p27), has been shown to participate in progestin-induced growth suppression of normal endometrial glands. To analyse the molecular mechanisms regulating p27 protein, we examined immunohistochemical expression of the SCF(Skp2) (Skp1-Cullin-F-box protein) complex factors, i.e. Skp1, Cul1 and Skp2, and compared them with that of p27, steroid receptors and Ki-67. In normal endometrial glands, the expression of Skp2 was observed in the proliferative phase, whereas that of p27 was observed in the secretory phase. Cultured normal endometrial glandular cells showed that progesterone induced the down-regulation of Skp2 along with up-regulation of p27. In endometrial carcinomas, the inverse topological correlation between Skp2 and p27 was evident in 39/66 (59%) cases, and the expression of Skp2 showed a strong correlation with Ki-67. These findings suggest that the expression of SCF(Skp2) complex changes during the menstrual cycle in normal endometrium and the SCF(Skp2) ubiquitin-proteasome pathway may also work in endometrial carcinomas.

Id1 Provides a Proper Hematopoietic Progenitor Niche Function

Development of hematopoietic stem cells (HSC) and their progeny is maintained by the interaction with cells in the microenvironment. In addition to hematopoietic cells, Id1 is expressed in stromal cells known to support hematopoiesis, and is involved in cell proliferation, differentiation and senescence. Therefore, to investigate the role of Id1 in hematopoiesis, we examined hematologic phenotypes of Id1−/− mice. In this study, we found increased neutrophils and macrophages, and decreased B cells and platelets in peripheral blood, and decreased BM cellularity. While the percentages of hematopoietic stem cells (HSC) in Id1−/− mice were increased relative to the Id1+/+ mice, their total numbers and function appeared normal. For example, Id1 was not required for self-renewal or repopulation of HSC. In contrast, we found that there were increased numbers of hematopoietic progenitor cells (HPC) in S phase of cell cycle in Id1−/− mice BM, suggesting that the loss of Id1 within HPC promotes proliferation. However, purified Id1−/− HPC had the same proliferation potential as Id1+/+ HPC when cultured in vitro. In transplantation experiments, we proved that BM microenvironment in Id1−/− mice is defective by showing that the Id1+/+ HSC showed impaired hematopoietic development in Id1−/− mice, while the Id1−/− HSC had normal repopulation potential in an Id1+/+ microenvironment. In agreement with these findings, Id1−/− BM stromal cell cultures supported enhanced proliferation of hematopoietic progenitors. Furthermore, quantitative PCR showed that SCF, M-CSF, OPN, SDF-1 and TGF-α mRNA expression was decreased in Id1−/− stromal cells relative to Id1+/+ stromal cells, while G-CSF, GM-CSF, and VEGF mRNA expression was significantly increased. Id1−/− BM showed decreased number of mesenchymal stem/progenitor cells. Thus, Id1 does not play a role in maintaining HSC, but is involved in regulating hematopoietic progenitor niche. Funded by NCI contract No. N01-CO-12400.

NF-κB and the nuclear kinase MSK1 in a murine model of asthma

We previously reported that NF-κB activation is essential for the overexpression of the major mast cell growth factor SCF in inflammation (Da Silva et coll., Faseb J 2003). We show here the activation of the nuclear kinase MSK1 as the key step of NF-kB activation, and the consequences in a murine asthma model. NF-κB p65 was phosphorylated at Ser276 upon IL-1β treatment as shown by Western blotting. Chromatin immunoprecipitation experiment showed binding of NF-κB, of MSK1 and of CBP to the SCF gene κB intronic enhancer. Further on, IL-1β upregulated SCF expression and this was abolished by S276C p65 mutation, MSK1 kinase-dead mutation, anti-MSK1 siRNA transfection, or by pharmacological inhibition by the H89 kinase inhibitor compound, indicating a role for MSK1 mediated S276 p65 phosphorylation in SCF expression. Additionally, we evaluated the effect of this kinase inhibitor, compound H89 (10 mg/kg), as compared to solvent in a murine asthma model created by sensitization and challenge of mice with ovalbumin + Al(OH)3. The eosinophilic infiltrate and the associated remodelling of the airways including mucus secretion were inhibited by 80% and 60%, respectively. In conclusion, we therefore propose MSK1 as an interesting therapeutic target to combat mast cell-associated inflammation in particular in asthma.

Differential regulation of human and mouse telomerase reverse transcriptase (TERT) promoter activity during testis development

In adult testis, telomerase activity is exclusively detectable during the early steps of spermatogenesis and is downregulated during differentiation to spermatozoa. Knowledge about telomerase activity during testis development from birth to adulthood is still scarce. Telomerase activity is regulated primarily via the transcriptional initiation of TERT expression which encodes its catalytic subunit. We used the hTERTp-lacZ transgenic mouse model that expresses the bacterial lacZ reporter gene under the control of an 8.0-kbp human TERT promoter fragment to analyze simultaneously endogenous mouse Tert gene expression as well as human TERT promoter activity during mouse testis development. We show that human TERT promoter activity increased during puberty and was highest in adult mouse testis whereas mouse Tert expression and telomerase activity were found to be high in testis from the earliest time point tested (6 days postpartum). Histological analysis revealed that beta-galactosidase expression, encoded by the lacZ reporter gene, is present in all seminiferous tubules in adult testis, but in a subset of tubules before puberty. We further analyzed the expression of SCF/ c-KIT, which was described to regulate spermatogonia proliferation and mouse Tert expression, in prepubertal and adult testis by immunohistochemistry. Whereas SCF and c-KIT were detectable in all seminiferous tubules, a spatial and preclusive expression pattern of human TERT promoter activity and activated c-KIT (p-c-KIT Tyr 567/569) was observed in prepubertal testes.

Change and significance of the expression of c-kit and SCF following recovery from unilateral testicular torsion in rats

To investigate the change in expression levels of c-kit and SCF, and the protective effects of FSH on ischemia-reperfusion injury due to testicular torsion-detorsion. 24 adult male SD rats were divided into three groups of 8: control group, testicular torsion group and FSH-treated group. The control group was treated with sham-operation. Animals in the testicular torsion and FSH-treated groups were subjected to unilateral 720 degrees counterclockwise testicular torsion for 2 hours and then reperfusion was allowed after detorsion. The FSH-treated group received intraperitoneal injection of FSH 15min before detorsion. Then, the rats were sacrificed and the testes were harvested. Histopathological changes were observed by light microscope, and the expression levels of c-kit, SCF in testicular tissue in the different groups were detected by Immunohistochemical assay and Quantitative Real-time RT-PCR analysis. Finally, the relative proportions of germ cells were measured by FCM. c-kit and SCF were positive expressed in 52.58% and 61.16% of testicular cells of control tissues, respectively. Decreases of c-kit and SCF positive cells (15.01% and 9.18%) were found in the testicular torsion group. After being treated by FSH, the number of positive cells increased (31.25% and 20.01%). Moreover, the c-kit and SCF mRNA expression was increased dramatically (P < 0.01) in response to FSH stimulation. Furthermore, the number of haploid, diploid and tetraploid cells has also increased significantly in drug-treated testes (P < 0.01). The mechanism of tissue damage in the testicular torsion model, includes changes in the expression of c-kit and SCF following torsion. Also, FSH has a protective effect on germ cells after unilateral testicular torsion, which was reflected by increased c-kit and SCF levels.

Hematopoietic Effect of Phellinus linteus Polysaccharide in Mouse Splenocytes and Bone Marrow Cells

In anti-cancer therapies, radiotherapy and chemotherapy show a superior inhibition effect on cancer cell growth, but those are very toxic to normal tissues and organs. Particularly, drugs for neutropenia, one of chemotherapy agents, which suppress the function of bone marrow, are needed to be controlled in terms of their dosage and therapy period because of their side effect. Phellinus linteus polysaccharide (PL) has been reported to increase the number of splenocytes and bone marrow cells. PL has been shown to decrease the side effects of cyclophosphamide (CYC) treatment to the cancer patients. PL showed no effects in semisolid clonogenic assay, suggesting that PL doesn't contain substantial compounds to substitute for colony stimulating factors (CSFs). On the other hand, PL increased the expression of SCF, IL-3, GM-CSF, TPO genes. These results indicate that PL may promote the growth and proliferation of splenocytes and bone marrow cells through indirect or CSFs-dependent pathway, which may lead to a hematopoiesis.

Kit-stem cell factor (kit ligand) axis in GIST patients treated with imatinib

10046 Background: Gastrointestinal stromal tumors (GISTs) represent the majority of GI mesenchymal neoplasms. Imatinib (Gleevec; STI571; Novartis, Basel, Switzerland) has marked efficacy in the management of GIST; however, little is known of its exact mechanism in GIST. We sought to elucidate expression patterns of stem cell factor (SCF, Kit ligand) and the Kit receptor tyrosine kinase in GIST and tumor-associated endothelial cells (TECs) from patients treated with imatinib. Methods: We constructed a 54 GIST specimen tissue array from patients with GIST surgically resected who received 400–800 mg/day adjuvant imatinib. Arrays were stained for SCF by immunohistochemistry and scored for expression as 0 (none), 1 (weak), or 2 (strong). Expression was correlated to progression-free survival (PFS) and overall survival (OS) using Kaplan-Meier methods. In a second study we collected pre-imatinib core needle biopsies from 15 patients on a phase II prospective, randomized clinical trial of neo-adjuvant and adjuvant imatinib (600mg/day) for the treatment of primary or advanced, resectable GIST. Specimens were stained for phosphorylated-Kit and CD31 and visualized by immunofluorescence and confocal microscopy. Co-localization of markers was correlated to radiographic response by positron emission tomography (PET) and dynamic computed tomography (dCT). Results: 47 of 54 tumors (87%) expressed SCF but there was no significant correlation between SCF expression or intensity with PFS and OS. Interestingly, the majority of GIST tissue from patients on the prospective clinical trial contained pre-imatinib TECs expressing phosphorylated-Kit. 69% and 79% of patients receiving 3–7 days of imatinib responded by PET scan or dCT, respectively. Conclusions: The majority of GISTs express SCF while pre-imatinib TECs were found to express phosphorylated-Kit. Therefore, our results suggest that within the tumor microenvironment there may be Kit/SCF crosstalk between tumor cells and TECs. Whether disruption of this crosstalk plays a role in tumor response or anti-vascular efficacy requires additional investigation. No significant financial relationships to disclose.

Enhanced expression of mast cell growth factor and mast cell activation in the bladder following the resolution of trinitrobenzenesulfonic acid (TNBS) colitis in female rats

Chronic pelvic pain disorders often overlap. We have shown that acute colonic irritation can produce acute irritative micturition patterns and acutely sensitize bladder afferent responses to mechanical and chemical stimuli. We hypothesize that with time, colonic irritation can lead to neurogenic changes in the bladder and the development of chronic bladder sensitization. Micturition patterns were measured in rats 60-90 days after the induction of trinitrobenzenesulfonic acid (TNBS) colitis in the resolution phase of this model. Total and activated mast cells (MCs) were quantified in the bladder, while mRNA levels of stem cell factor (SCF; a.k.a. MC growth factor) and nerve growth factor (NGF; a MC and nociceptive C-fiber stimulator) were quantified in the bladder and L6-S1 dorsal root ganglia (DRG). Following intra-rectal TNBS, voiding volume was reduced (P < 0.005), while voiding frequency was increased (P < 0.05), both by approximately 50%. Furthermore, both the percentage and density of activated bladder MCs were significantly elevated (P < 0.05), although total MC counts were not statistically increased. At the molecular level, urinary bladder SCF expression increased twofold (P < 0.005), as did NGF (P < 0.01), while L6-S1 DRG levels were not significantly elevated. Chronic cystitis in the rat as evidenced by changes in micturition patterns and the recruitment of activated MCs can occur during the resolution phase of TNBS colitis. These changes, of which MCs may play an important role, appear to be maintained over time and may occur via stimulation of convergent pelvic afferent input resulting in the upregulation of neurotrophic factors in the target organ.

Stem cell-related cardiac gene expression early after murine myocardial infarction

Clinical experimental stem cell therapy after myocardial infarction appears feasible, but its use has preceded the understanding of the working mechanism. The ischemic recipient cardiac environment is determinative for the attraction and subsequent fate of stem cells. Here, we studied expression levels of genes that are anticipated to be essential for adequate stem cell-based cardiac repair at various time-points during the 1 month period following myocardial infarction (MI). Gene expression in the hearts of mice that underwent MI by permanent or transient (30 min) ligation of the coronary artery was monitored using quantitative RT-PCR analysis of mRNA isolated from whole heart sections as well as from specific, laser micro-dissected, regions of sections. Protein expression was performed by immunohistochemical stainings and Western blot analysis. Many inflammatory genes were highly expressed for at least 1 week after MI. The expression of pro-angiogenic genes such as bFGF, VEGF-A and VEGF-R2 changed only marginally post-MI. Markers used to test stem cell gene expression remained unchanged post-MI with the exception of G-CSF and GM-CSF, which are genes that are also known to enhance the inflammatory response. Analysis of micro-dissected regions revealed that SDF-1, SCF (both stem cell attractants) and VEGF-R2 (involved in angiogenesis) gene expression was slightly decreased especially in the infarcted region. Genes that are generally considered to participate in stem cell-related processes and angiogenesis were not upregulated after MI, whereas the inflammatory gene expression dominated. Modulation of this imbalance might be of value for stem cell-mediated therapy.

Human amniotic fluid‐derived stem cells have characteristics of multipotent stem cells

To characterize mesenchymal stem cell-like cells isolated from human amniotic fluid for a new source of therapeutic cells. Fibroblastoid-type cells obtained from amniotic fluid at the time of birth. The ability of ex vivo expansion was investigated until senescence, and stem cell-like characteristics were analyzed by examining differentiation potential, messenger RNA expression and immunophenotypes. A morphologically homogenous population of fibroblastoid-type (HAFFTs) cells, similar to mesenchymal stem cells from bone marrow (BM-MSCs), was obtained at the third passage. The cells became senescent after 27 passages over a period of 8 months while undergoing 66 population doublings. Under appropriate culture conditions, by the 8th passage they differentiated into adipocytes, osteocytes, chondrocytes and neuronal cells, as revealed by oil red O, von Kossa, Alcian blue and anti-NeuN antibody staining, respectively. Immunophenotype analyses at the 17th passage demonstrated the presence of TRA-1-60; SSEA-3 and-4; collagen types I, II, III, IV and XII; fibronectin; alpha-SMA; vimentin; desmin; CK18; CD44; CD54; CD106; FSP; vWF; CD31; and HLA ABC. Reverse transcriptase-polymerase chain reaction analysis of the HAFFTs from passages 6-20 showed consistent expression of Rex-1, SCF, GATA-4, vimentin, CK18, FGF-5 and HLA ABC genes. Oct-4 gene expression was observed up to the 19th passage but not at the 20th passage. HAFFTs showed telomerase activity at the 5th passage with a decreased level by the 21st passage. Interestingly, BMP-4, AFP, nestin and HNF-4alpha genes showed differential gene expression during ex vivo expansion. Taken together, these observations suggest that HAFFTs are pluripotent stem cells that are less differentiated than BM-MSCs, and that their gene expression profiles vary with passage number during ex vivo expansion.

Expression of C-kit and SCF in human cholangiolocellular carcinoma and its significance

目的 拟通过检测C-kit、SCF基因在胆管细胞癌中的表达来了解C-kit、SCF与胆管细胞癌发生和发展之间的关系.方法采用免疫组化S-P法检测48例肝胆管细胞癌及20例肝外胆管癌标本C-kit、SCF蛋白表达情况,同时取对应的癌旁胆管组织及另取30例肝及胆管良性病变胆管组织作对照,所有数据采用x2检验.结果48例肝胆管细胞癌组织中C-kit、SCF蛋白表达分别为39例(81.3%)和36例(75.0%),20例肝外胆管癌组织中C-kit、SCF蛋白表达分别为12例(60.0%)和13例(65.0%),癌旁胆管组织中4例C-kit、5例SCF呈弱阳性表达,30例肝及胆管良性病变胆管组织C-kit、SCF蛋白均呈阴性表达.两癌组织与癌旁胆管组织、肝及胆管良性病变胆管组织C-kit、SCF表达阳性率比较差异有显著性(P＜0.05).Ⅲ～Ⅳ期胆管细胞癌C-kit、SCF蛋白表达阳性率高于Ⅰ～Ⅱ期(P＜0.05),中低分化癌高于高分化癌(P＜0.05),有淋巴结转移癌高于无淋巴结转移癌(P＜0.05).而肝外胆管癌组织中C-kit、SCF蛋白表达与其临床病理各因素无明显相关(P＞0.05).C-kit、SCF蛋白表达与胆管细胞癌病人年龄、性别、肿瘤直径无关.结论C-kit、SCF蛋白过表达可能在胆管细胞癌的发生、发展中发挥作用。

Indispensable role of Bcl2 in the development of the melanocyte stem cell

Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2.

Interleukin-4 and 13 Induce the Expression and Release of Monocyte Chemoattractant Protein 1, Interleukin-6 and Stem Cell Factor From Human Detrusor Smooth Muscle Cells: Synergy With Interleukin-1β and Tumor Necrosis Factor-α

Interstitial cystitis is characterized by an increased number of activated MCs in the detrusor muscle. However, to our knowledge the factors that influence the anatomical relationship between MCs and HDSMCs are unknown. MCP-1, IL-6 and SCF have a critical role in the regulation of MC development, signaling and function. We investigated whether HDSMCs are capable of expressing and releasing MCP-1, IL-6 and SCF in response to IL-4, IL-13, IL-1beta and tumor necrosis factor-alpha. HDSMCs were isolated and cultured using an explant technique. Protein expression, and the secretion of MCP-1, IL-6 and SCF were assayed by semiquantitative reverse transcriptase-polymerase chain reaction and specific enzyme-linked immunosorbent assay. Unstimulated cells released low amounts of MCP-1, IL-6 and SCF. In cells stimulated by IL-4 MCP-1 mRNA was up-regulated by a mean factor +/- SD of 3.5 +/- 1.3, IL-6 mRNA was up-regulated by 3.8 +/- 1.3, the soluble form of SCF was up-regulated by 3.2 +/- 0.6 and the membrane bound form of SCF was up-regulated by 7.9 +/- 5.6. For IL-13 stimulated cells the values were 2.6 +/- 1.5, 3.6 +/- 2.1, 2.9 +/- 1.6 and 5.7 +/- 3.7, respectively. Soluble SCF mRNA expression was 5 times higher than the expression of mSCF mRNA. IL-4 and IL-13 given separately stimulated MCP-1, IL-6 and SCF secretion in a concentration (0.01 to 100 ng/ml) and time (0 to 24 hours) dependent manner. Furthermore, IL-1beta and tumor necrosis factor-alpha alone induced significant release of MCP-1, IL-6 and SCF but in combination with IL-4 or IL-13 it induced greater secretion of MCP-1, IL-6 and SCF. To our knowledge these findings demonstrate for the first time that HDSMCs express and release MCP-1 and IL-6, and show relatively high expression of soluble SCF. This supports our hypothesis that HDSMCs may have an active role by orchestrating the local inflammatory response in the bladder wall with possible implications for the pathophysiology of detrusor mastocytosis.

Murine serum obtained from bone marrow-transplanted mice promotes the proliferation of hematopoietic stem cells by co-culture with MS-5 murine stromal cells

To examine whether serum obtained from bone marrow-transplanted mice can selectively expand hematopoietic stem cells (HSCs) among whole bone marrow cells in vitro, whole bone marrow cells were cultured with or without MS-5 murine stromal cells in the presence of serum obtained from transplanted mice on day 3 (day 3 serum) or serum from normal mice for 7 days. When whole bone marrow cells and MS-5 cells were co-cultured in day 3 serum for 7 days, the c-kit-positive, Sca-1-positive, lineage marker-negative cells (KSL cells) expanded approximately 25 times; however, when they were co-cultured in normal serum for 7 days, the KSL cells expanded approximately 1.3 times. Direct contact between the whole bone marrow cells and MS-5 cells was essential for expansion of KSL cells in the co-culture, and it upregulated the expression of some cytokines in MS-5. Above all, the day 3 serum specifically upregulated the expression of SCF, SDF-1 alpha, G-CSF, IL-11 and IL-6 in MS-5. The level of testosterone in the day 3 serum was higher than normal serum and the addition of the testosterone in the culture expanded the KSL cells among whole bone marrow cells on MS-5 cells and also upregulated the expression of SDF-1 alpha, IL-11 and IL-6 in MS-5. These data indicates that the serum of bone marrow-transplanted mice contains a factor(s) that induced changes in the expression levels of various cytokines in MS-5 stromal cells and enabled the MS-5 cells to expand the KSL cells among whole bone marrow cells.

Properties of MSC Derived Stromal Cell Lines Developed from Bone Marrow of TNF Knockout Mice.

Mesenchymal stem cells (MSC) forming adherent stromal cell lines have been revealed in suspension fraction (SF) of 70+ weeks old long-term bone marrow culture (LTBMC) of TNF knockout mice.The proportion of CD45+ cells in such old cultures decreased significantly while cellularity of SF increased, accompanied by the first successful generation of cell lines. More than 100 cell lines were established.Almost all cell lines were Sca1 positive, expressed SDF1, had fibroblast-like morphology and expressed collagens type II and IV. Most of cell lines could maintain hematopoiesis (CFU-S, CFU-GM and CAFC production) for 10 weeks. Some of the cell lines were able to maintain growth without differentiation of murine ES cells and some expressed LIF or LIF-receptor, VEGF and FGF1. All cell lines produced hematopoietic growth factors - their supernatants stimulated growth of CFU-GM in semisolid media. The expression of SCF was revealed in all cell lines tested for growth factors production.Quantitative RT-PCR analysis revealed that the expression of hematopoietic growth factors - M-CSF, G-CSF and TPO increased sometimes up to 6 times in comparison with the expression of these factors in adherent cell layer (ACL) of LTBMC. The expression of N-cadherin - one of the main regulators of hematopoietic stem cells (HSC) in niche - increased 7–32 fold in 7 tested cell lines regarding the expression of this RNA in ACL of LTBMC. Whereas the expression level of N-cadherin was lower in wild type bone marrow, NIH3T3 fibroblasts and MS5 cells, maintaining the hematopoiesis and CAFC growth, showed the expression on the higher level than in ACL. The expression of ICAM 1, the molecule also regulating HSC, was reduced in all cell lines. The expression level of genes participating in activating of hematopoietic stem cells in niche such as Jagged, Dll 1 and Ang1 more than halved in 5 out of 7 tested lines. The production of Wnt 5a RNA fluctuated from 0,5 to 12 fold and did not correlate with any other changes in gene expression and the ability of cell lines to maintain hematopoiesis. The level of Ihh, Shh was downregulated in cell lines.The cell line 26dD3 developed in the media containing dexamethasone demonstrated 70 fold increased expression of Kirre - gene supporting HSC but there was no difference in maintaining hematopoiesis and ES cells. High expression of human homolog of this gene (NEPH2) was known to be detected in brain only. At the same time this cell line expressed tubulin b3. These data support a model of lineage specification in which unilineage commitment is prefaced by a “promiscuous” phase of multilineage locus activation. Thus the developed stromal cell lines are not terminally differentiated and use different pathways to maintain hematopoiesis. The TNF deficiency leads to dramatic alterations in regulation of MSC. Mechanisms of this regulation should be scrutinized.

Biostimulation of dermal fibroblast by sublethal Q-switched Nd:YAG 532 nm laser: Collagen remodeling and pigmentation

The application of medical lasers in treating pigmented lesions has rapidly developed over the past decade. In both clinical and cosmetic application, melanin is targeted in pigmented areas and destroyed by the mechanism of selective photothermolysis. When laser radiation passes through superficial pigmented tissue, energy will be further reduced by dermal collagen scattering and absorption. Non-pigmented dermal fibroblasts will be exposed to co-incidental laser irradiation at lower energy levels. Biostimulation of dermal fibroblasts by low energy laser is reported in this paper. The Q-switched frequency doubled Nd:YAG 532 nm laser used in clinical laser therapy was used in this study. Sublethal laser fluence was determined at 0.8 J/cm 2 and used to stimulate normal human fibroblasts in monolayer culture. The results showed that there was no significant difference in collagen synthesis between the stimulated fibroblasts and controls. However, significant delay in collagen remodeling activity was demonstrated in the irradiated group by measuring fibroblast populated collagen lattice (FPCL) contraction. The stimulation of SCF, HGF and b-FGF gene expression was determined by RT-PCR analysis and demonstrated to vary between cases. Two out of six cell lineages that showed stronger responses to laser stimulation on SCF, HGF and b-FGF gene expressions were used to prepare conditioned media. The conditioned media from irradiated groups showed significant increase in SCF and b-FGF content and stimulated SK-mel-3 melanoma cells to synthesize more melanin in vitro. These results suggest that sublethal laser stimulation of fibroblasts may cause post-laser hyperpigmentation through production of melanogenic stimulatory cytokines. The degree of stimulation of SCF, HGF and b-FGF production varied between individual cell lineages, which may reflect the true variation of post-laser hyperpigmentation in clinical practice.

Molecular Characterization of PDGFR-α/PDGF-A and c-KIT/SCF in Gliosarcomas

Gliosarcomas are rare and poorly characterized malignant brain tumors that exhibit a biphasic tissue pattern with areas of gliomatous and sarcomatous differentiation. These tumors are histological variants of glioblastoma, displaying a similar genetic profile and dismal prognosis. Up-regulation of PDGFR subfamily of tyrosine kinase members, PDGFR-α and c-Kit, and their intracellular effectors RAS/RAF/MAPK has a crucial role in the cancer development. In addition, signal transduction mediated by activating mutations of c-Kit and PDGFR can be effectively blocked by specific tyrosine kinase inhibitors, such as Imatinib mesylate. The aim of this study was to characterize the molecular alterations of PDGFR signaling in gliosarcomas. Six cases were analyzed by immunohistochemistry for the expression of PDGFR-α, c-Kit and their ligands PDGF-A and SCF, respectively. The cases were further evaluated for the presence of activating mutations of PDGFR-α (exons 12 and 18) and c-kit (exons 9, 11, 13, and 17), as well as B-RAF (exons 11 and 15). Expression of PDGF-A was found in all cases and co-expression of PDGFR-α was observed in three cases. Four cases showed expression of SCF, and c-Kit was observed only in one case that also expressed SCF. Generally, immunoreaction predominates in the glial component. The mutational analysis of PDGFR-α showed the presence of an IVS17-50insT intronic insertion in two cases, one of them also with a 2472C > T silent mutation; this silent mutation was also found in another case. Glioma cell line analysis of IVS17-50insT insertion showed no influence on PDGFR-α gene splicing. No mutations were detected in c-kit and B-RAF oncogenes. Our Results indicate that activating mutations of PDGFR-α, c-kit and B-RAF are absent in gliosarcomas. Nevertheless, the presence of a PDGFR-a/PDGFA and c-Kit/SCF autocrine/paracrine stimulation loop in a proportion of cases, supports the potential role of specific tyrosine kinase inhibitors in the treatment of gliosarcomas.

Stro-1 Positive and Stro-1− Negtive Human Mesenchymal Stem Cells Express Different Levels of Immunosuppression.

Mesenchymal stem cells (MSCs), have been shown to elicit immunosuppressive effect on allogeneic lymphocyte response. However, MSCs are heterogeneous and data on the inhibitory abilities of different MSC subsets are lacking. In the present study, we selected Stro-1+ cells from human bone marrow and evaluated the inhibitory capability of this MSC subset in mixed lymphocyte reactions (MLRs) or in mitogen stimulation asssays, in comparison to that of Stro-1− cells. To evaluate the two MSC subsets for immunomodulation in vitro, we added 1,000–30,000 Stro-1+ or Stro-1− cells to MLR at the beginning of the experiment. When comparing the inhibitory effects of the two subsets, PBLs proliferation was significantly more inhibited by Stro-1+MSCs (11.0%–63.7%) than by stro-1−MSCs (35.5%-106%) (P<0.01). Furthermore, as few as 1,000 Stro-1+ MSC could inhibit lymphocyte proliferation more effectively than 10 times more (10,000 cells) Stro-1−cells. As it was observed with the mixed lymphocyte reaction, suppression of the response to the mitogen also occurred in a dose dependent fashion, but to a lesser extent with the Stro-1−cells (25.5%–80.1% vs 7.5%–38.4% in Stro-1+cells) (P<0.05). To investigate whether the difference of suppressive effect that we observed between Stro-1+ and Stro-1− cells, still exist when MSC subsets are separated physically from PBL, we performed MLR in the upper chamber of a transwell and we seeded the lower chamber either with Stro-1+ or Stro-1− cells. The inhibitory effect of Stro-1+ cells was significantly more profound than the one observed when Stro-1− cells were used in the Transwell culture system (p<0.05) (Figure 3), demonstrating that one or several soluble factors was involved in production of different suppressive effects. Cytokine and chemokine genes, IL-10, TGF-β1, SDF-1, SCF and IL-6 expression were evaluated in both MSC subsets by quantitative RT-PCR. Low levels of IL-6, SCF, SDF-1 were observed in Stro-1+, which induced a fold increase around 1 (0,96 ± 0,32; 0,96 ± 0,24; 0,96 ± 0,24), indicating that there is no signifiant difference of these genes expression between the two MSC subsets. However, we observed in Stro-1+ a decreased gene expression for IL-10 (0,24 fold ± 0,59; p <0,05) and for TGF b1 (0,43 fold ± 0,32; p <0,05). This finding suggested that the candidate T-cell inhibitory factors TGF b1, IL-10, which are lower expressed in Stro-1+ cells, are not responsible for the more profound inhibition of immunoreactivity by Stro-1+ cells. We show here that significant differences do exist within these two subsets. Stro-1+ cells inhibit lymphocyte proliferation significantly more profoundly than Stro-1−cells. The difference is in part mediated by soluble factors, but not IL-10 and TGF-β1. These results point to the notion that Stro-1+ cells can elicit more powerful immunosuppressive ability and a pre-selection of Stro-1+MSC for clinical use may be advisable. These findings suggest that pre-selection of MSC before clinical use might produce more effective immunosuppression in different therapeutic applications, especially in clinics for the prevention of graft versus host disease (GVHD).