Proliferation-promoting Factors Research Articles

Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells

BackgroundLiver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function.MethodsHuman PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses.ResultsGStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation.ConclusionsWe generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.

ヒト膵β細胞増殖促進因子の探索

ヒト膵β細胞増殖促進因子の探索

Molecular tumor board-renal cell carcinoma

The introduction of molecular targeted agents has fundamentally changed the treatment of metastatic renal cell carcinoma. Afirst wave of development was based on the improved understanding of tumor biology since the discovery of the importance of the von Hippel-Lindau gene as the key driver of the disease and paved the way for antiangiogenic agents. Of relevance is the overexpression of proangiogenic and proliferation-promoting factors (VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor) as well as an overactivation of the PI3K-Akt signaling pathway: the target structure is the "mammalian target of rapamycin" (mTOR) molecule, which is involved in the regulation of cell proliferative processes. VEGF-, PDGF-, and mTOR-signals and signaling pathways are central targets of current targeted substances. Asecond wave is certainly to be seen in the development of therapeutic approaches with the targeted activation and modulation of the immune system, which has brought "immunotherapy" back into the focus of interest. Central development is the application of immune-checkpoint inhibitors, with the help of which (re-)activation of the cellular defense, especially of Tcells, takes place, which perse holds the potential of acytoreductive therapy by killing the tumor cells. Even though the prognosis has improved significantly due to the rapid development of recent years, treatment remains challenging as most patients experience progress, and long-term survival is only achieved in about 20% of cases because some patients are primarily refractory or do not respond. The more intensive interlocking of molecular biology, pathology, clinical research, and interdisciplinary uro-oncology, as is the claim of molecular tumor boards, can contribute to the individual selection of asuitable therapy strategy and, thus, establish the latest findings and developments for the benefit of patients in the clinic.

Lipid core nanoparticles as vehicle for docetaxel reduces atherosclerotic lesion, inflammation, cell death and proliferation in an atherosclerosis rabbit model

Chemotherapeutic agents used in cancer treatment associated to nanoparticles (LDE) that mimic the composition of low-density lipoprotein and buffer their toxicity can have strong anti-atherosclerosis action, as we showed in cholesterol-fed rabbits. Here, a novel preparation of docetaxel (DTX) carried in LDE was evaluated. Eighteen rabbits were fed 1% cholesterol during 8 weeks. After the first 4 weeks, 9 animals were treated for 4 weeks with intravenous LDE-DTX (1 mg/kg/week) and 9 with LDE only (controls) once a week for 4 weeks. Animals were then euthanized and the aortas were analyzed for morphometry, immunohistochemistry and Western blot. LDE-DTX treated group showed 80% reduction of atheroma area compared to controls. LDE-DTX treatment reduced in 60% the protein expression of macrophage marker CD68 and of MCP-1 in 80%. LDE-DTX pronouncedly lowered expression of pro-inflammatory markers NF-κB, TNF-α, IL-1β, IL-6 and von Willebrand factor and elicited 40% reduction in cell proliferation marker PCNA. The presence of smooth muscle cells in the intima was 85% smaller than in controls. Pro-apoptotic caspase 3, caspase 9, Bax, and anti-apoptotic Bcl-2 all were reduced by LDE-DTX. Protein expression of MMP-2 and MMP-9, TGF-β, and collagen 1 and 3 were also markedly lowered by the LDE-DTX treatment. Animals showed no hematological, hepatic or renal toxicity consequent to LDE-DTX treatment. In conclusion, LDE-DTX showed a wide array of strong effects on pro-inflammatory and proliferation-promoting factors that drive the lesion development. These findings and the lack of observable toxicity indicate that LDE-DTX can be a candidate for future clinical trials.

Disrupting LXRα phosphorylation promotes FoxM1 expression and modulates atherosclerosis by inducing macrophage proliferation

Macrophages are key immune cells for the initiation and development of atherosclerotic lesions. However, the macrophage regulatory nodes that determine how lesions progress in response to dietary challenges are not fully understood. Liver X receptors (LXRs) are sterol-regulated transcription factors that play a central role in atherosclerosis by integrating cholesterol homeostasis and immunity. LXR pharmacological activation elicits a robust antiatherosclerotic transcriptional program in macrophages that can be affected by LXRα S196 phosphorylation in vitro. To investigate the impact of these transcriptional changes in atherosclerosis development, we have generated mice carrying a Ser-to-Ala mutation in myeloid cells in the LDL receptor (LDLR)-deficient atherosclerotic background (M-S196ALdlr-KO). M-S196ALdlr-KO mice fed a high-fat diet exhibit increased atherosclerotic plaque burden and lesions with smaller necrotic cores and thinner fibrous caps. These diet-induced phenotypic changes are consistent with a reprogramed macrophage transcriptome promoted by LXRα-S196A during atherosclerosis development. Remarkably, expression of several proliferation-promoting factors, including the protooncogene FoxM1 and its targets, is induced by LXRα-S196A. This is consistent with increased proliferation of plaque-resident cells in M-S196ALdlr-KO mice. Moreover, disrupted LXRα phosphorylation increases expression of phagocytic molecules, resulting in increased apoptotic cell removal by macrophages, explaining the reduced necrotic cores. Finally, the macrophage transcriptome promoted by LXRα-S196A under dietary perturbation is markedly distinct from that revealed by LXR ligand activation, highlighting the singularity of this posttranslational modification. Overall, our findings demonstrate that LXRα phosphorylation at S196 is an important determinant of atherosclerotic plaque development through selective changes in gene transcription that affect multiple pathways.

The Proliferation Capacity of Cultured Neural Stem Cells Promoted by CSF Collected from SAH Patients Correlates to Clinical Outcome

Neurogenesis from endogenous neural stem cells (NSCs) might contribute to functional recovery after stroke based on animal studies; however, the relationship between neurogenesis and post-stroke outcome has rarely been demonstrated in humans. We prospectively collected cerebrospinal fluid (CSF) from 36 patients with subarachnoid hemorrhage (SAH). The CSF was added to the culture medium of the rat NSCs to test the effects on proliferation (proliferation index [PI], percentage of Ki-67 immunoreactive cells). We correlated the PI with functional outcome based on the modified Rankin Scale at 3 months post-SAH. Treatment with the CSF samples collected from SAH patients showed a higher PI compared with those collected from patients with normal pressure hydrocephalus and untreated controls (20.3 ± 8.8 vs. 8.2 ± 5.1 and 7.8 ± 3.0, P < 0.001), indicating proliferation-promoting factors in CSF after SAH. The PI was positively correlated with SAH volume (p = 0.025). For patients with lower SAH volume, patients with favorable outcome had a higher PI than those with poor outcome (20.8 ± 6.9 vs. 14.6 ± 4.3, p = 0.047). Using multivariable logistic regression analysis, the PI was a positive determinant for favorable outcome (odds ratio, 1.17; 95% confidence interval, 1.00 to 1.36) that more proliferation-promoting factors in CSF was associated with better functional outcome in SAH patients.

Age-dependent human β cell proliferation induced by glucagon-like peptide 1 and calcineurin signaling.

Inadequate pancreatic β cell function underlies type 1 and type 2 diabetes mellitus. Strategies to expand functional cells have focused on discovering and controlling mechanisms that limit the proliferation of human β cells. Here, we developed an engraftment strategy to examine age-associated human islet cell replication competence and reveal mechanisms underlying age-dependent decline of β cell proliferation in human islets. We found that exendin-4 (Ex-4), an agonist of the glucagon-like peptide 1 receptor (GLP-1R), stimulates human β cell proliferation in juvenile but not adult islets. This age-dependent responsiveness does not reflect loss of GLP-1R signaling in adult islets, since Ex-4 treatment stimulated insulin secretion by both juvenile and adult human β cells. We show that the mitogenic effect of Ex-4 requires calcineurin/nuclear factor of activated T cells (NFAT) signaling. In juvenile islets, Ex-4 induced expression of calcineurin/NFAT signaling components as well as target genes for proliferation-promoting factors, including NFATC1, FOXM1, and CCNA1. By contrast, expression of these factors in adult islet β cells was not affected by Ex-4 exposure. These studies reveal age-dependent signaling mechanisms regulating human β cell proliferation, and identify elements that could be adapted for therapeutic expansion of human β cells.

GLD-4-mediated translational activation regulates the size of the proliferative germ cell pool in the adult C. elegans germ line.

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.

Pro-angiogenic impact of dental stem cells in vitro and in vivo

Within the field of dental tissue engineering, the establishment of adequate tissue vascularization is one of the most important burdens to overcome. As vascular access within the tooth is restricted by the apical foramen, it is of major importance to implement effective vascularization strategies in order to recreate viable components of teeth and periodontal tissues. However, while the current regenerative approaches focus on the use of dental stem cells (DSCs), little is known about these cells and their ability to promote angiogenesis. Therefore, the present study aimed to elucidate the paracrine angiogenic properties of postnatal DSCs, in particular dental pulp stem cells (DPSCs), stem cells from the apical papilla (SCAPs) and dental follicle precursor cells (FSCs). An antibody array, together with RT-PCR and ELISA, pointed out the differential expression of pro-angiogenic as well as anti-angiogenic factors by cultured DSCs and human gingival fibroblasts (HGF-1). Despite the secretion of proliferation-promoting factors, DSCs caused no notable increase in the proliferation of human microvascular endothelial cells (HMEC-1). With regard to other aspects of the angiogenic cascade, DPSCs, SCAPs and HGF-1 significantly promoted endothelial migration in a transwell migration assay. DPSCs also had a pronounced effect on endothelial tubulogenesis, as was shown by an in vitro Matrigel™ assay. In the last part of this study, a chorioallantoic membrane assay demonstrated a sustained pro-angiogenic impact of DPSCs and SCAPs in an in vivo setting. Collectively, these data indicate a predominant pro-angiogenic influence of DPSCs and SCAPS in vitro and in vivo in comparison to FSCs, suggesting that both stem cell populations could potentially promote the vascularization of regenerated dental tissues.

Deficient leukemia inhibitory factor signaling in muscle precursor cells from patients with type 2 diabetes

The cytokine leukemia-inhibitory factor (LIF) is expressed by skeletal muscle and induces proliferation of muscle precursor cells, an important feature of skeletal muscle maintenance and repair. We hypothesized that muscle precursor cells from patients with type 2 diabetes had a deficient response to LIF. The mRNA and protein expressions of LIF and its receptor (LIFR) were measured in skeletal muscle biopsies from healthy individuals and patients with type 2 diabetes by use of qPCR and Western blot. LIF signaling and response were studied following administration of recombinant LIF and siRNA knockdown of suppressor of cytokine signaling (SOCS)3 in myoblast cultures established from healthy individuals and patients with type 2 diabetes. Myoblast proliferation rate was assessed by bromodeoxyuridine incorporation. LIF and LIFR proteins were increased in both muscle tissue and cultured myoblasts from diabetic patients. Nonetheless, in the diabetic myoblasts, LIF-induced phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 was impaired. The deficient response to LIF administration in the diabetic myoblasts was further emphasized by a lack of increase in LIF-stimulated cell proliferation and a decreased LIF-stimulated induction of the proliferation-promoting factors cyclin D1, JunB, and c-myc. SOCS3 protein was upregulated in diabetic myoblasts, and knockdown of SOCS3 rescued LIF-induced gene expression in diabetic myoblasts, whereas neither STAT1 or STAT3 signaling nor proliferation rate was affected. In conclusion, although LIF and LIFR proteins were increased in muscle tissue and myoblasts from diabetic patients, LIF signaling and LIF-stimulated cell proliferation were impaired in diabetic myoblasts, suggesting a novel mechanism by which muscle function is compromised in diabetes.

Heterogeneous cross-talk of E2F family members is crucially involved in growth modulatory effects of interferon-γ and EGF

There is growing evidence that deregulation of E2F transcription factors is causatively involved in the patho-physiology of various tumors. However, no data on the role of E2F family members in tumor biology of ovarian cancer are available. We here describe an expression study of all known E2F transcription factors and their co-activators DP-1 and DP-2 in various human ovarian cancer cell lines and the breast cancer cell line T47D and their involvement in pathways affected by interferon-g and EGF. A significant over-expression of the proliferation-promoting E2F1 and especially E2F2 points to a pivotal role in modulating the uncontrolled proliferation in ovarian cancer cells. Of special note is the fact that interferon-g treatment did not only caused a reduction of the proliferation-promoting transcription factors E2F1 and E2F2, but also increased the inhibiting transcription factors E2F4 and E2F5, thus underlining the importance of an E2F cross-talk in the anti-proliferative function of interferon-g. Moreover, an increase in DP-1 is probably involved in the proliferation-enhancing effect of EGF. Our study provides a new insight in the crucial role of E2F cross-talk, especially the role of the inhibiting transcription factors E2F4 and E2F5, in the tumor biology of cancer and its possible usefulness as targets in anti-cancer therapy.

Gamma interferon down-regulates Fer and induces its association with inactive Stat3 in colon carcinoma cells.

Gamma interferon (IFN-gamma) is a regulator of cell growth, which suppresses the proliferation of HT-29 colon carcinoma cells. Here we show that in HT-29 cells IFN-gamma transiently increased the cellular level of the tyrosine kinase Fer, whose functioning was found to be essential for the proliferation of malignant cell-lines. The transient elevation in the level of Fer, was followed by its down-regulation, an effect which was most prominent after 6-8 h of IFN-gamma treatment. Up- and down-regulation of Fer was paralleled by the activation and subsequent deactivation of Stat3, which is a potent oncogene and a putative substrate of the tyrosine kinase Fer. Moreover, IFN-gamma induced the association of Fer and Stat3 and the newly formed complex was most stable at the down-regulated states of the two proteins. Formation of the Fer/Stat3 complex was accompanied by an attenuation in cell-cycle progression and accumulation of cells in the G1 phase. Thus, Fer and Stat3 are two proliferation-promoting factors whose down-regulation could contribute to the cytostatic activity of IFN-gamma in colon carcinoma cells.

Cyst Fluids of Malignant Human Brain Tumors Contain Substances That Stimulate the Growth of Cultured Human Gliomas of Various Histological Type

Abstract The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.

Cyst fluids of malignant human brain tumors contain substances that stimulate the growth of cultured human gliomas of various histological type.

The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration. These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases). TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included. These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture. The presence of proliferation-promoting factors in all cyst fluids could be demonstrated. On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation. The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media. Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs.

Relation of Nucleotides and Nucleosides to Proliferation-Promoting Factors Produced by Ultra-Violet Irradiated Yeast Cells

RECENT reports by Loofbourow1 on the role of adenine nucleotides in wound hormone preparations from ultra-violet treated yeast cells prompt us to give a preliminary account of some of our experiments which bear upon the problem.

Proliferation-promoting Activities of Extracts from Ultra-violet Injured Yeast Cells and of Bios Components

NUMEROUS papers from these laboratories have dealt with the production of proliferation-promoting factors ('intercellular wound hormones') by cells subjected to various injuring agents. It seems warranted to believe that these substances are produced by living, injured cells as a response to injury and are not simply dead-cell disintegration products1. The question of the nature of these materials is of great interest. It might be assumed that they are merely normal growth substances produced or released in greater quantities under the conditions of injury, or it is possible that they represent specific 'injury substances'. Two lines of inquiry have been undertaken to answer the question: (1) chemical and spectrographic studies, which suggest a nucleic acid-like nature for the wound hormones and which definitely show that substances of this type are produced in greater quantity as a result of injury2 ; (2) comparative growth studies, on the wound hormone preparations and known growth substances. The present note is concerned with the latter.

Relation of Aeration to the Activity of Proliferation-Promoting Factors from Injured Cells

IN earlier tests of the proliferation-promoting activity of materials from injured cells (‘intercellular wound hormones’) in which yeast was used as the test organism1, the test cultures were grown in rocker tubes with continuous shaking2. This subjected them to appreciable aeration. In recent investigations of wound hormones, yeast was again employed as the test organism, but was cultured in stationary, cotton-plugged tubes suspended in a water bath. At intervals of several hours, the tubes were removed from the water bath, shaken, and the yeast population determined with a photo-electric densitometer. In these later experiments, all preparations failed to show appreciable activity, though they had been prepared strictly in accordance with previously used techniques1.

Production of Intercellular Hormones by Mechanical Injury

PREVIOUS papers from this laboratory have demonstrated the production of proliferation-promoting factors (intercellular wound hormones) by cell aggregates subjected to lethal ultra-violet or X-rays1, heteroauxin2, and carbon dioxide3. These factors are evidently produced by living cells as a response to injury, since the maximum production of the hormones precedes the maximum rate of killing3.

Chemical Nature of Proliferation-Promoting Factors from Injured Cells

WE have published investigations indicating that yeast1 and animal cells2 injured by ultra-violet light and other means produce factors ('intercellular wound hormones')3 which are apparently released by injured, but living, cells into the intercellular fluid and stimulate the proliferation of normal cells. Spectrographic and chemical studies indicate that the active materials from yeast contain nucleic acid derivatives, as previously suggested4.