Culture-expanded human dermal stem cells exhibit donor to donor differences in cAMP generation

Stem cell techniques have facilitated a number of potential uses for such cells in cell therapy and drug development. Studies of the cAMP/protein kinase A (PKA) pathway are widely employed to investigate the effects of a large variety of substances. We assayed the cAMP pathway in human skin-derived mesenchymal stem cells (S-MSC) to evaluate donor to donor variations in response to pharmacological manipulations in vitro. Immunophenotyping of S-MSC revealed that, in general, 95% of S-MSCs were positive for CD90, CD73 and CD105 and negative for the expression of haemopoetic markers CD14, CD45 and human leukocyte antigen-DR (HLA-DR). Nevertheless, fluctuations occurred in basal cAMP levels from 5 pmol/mg to 18 pmol/mg. Total cAMP response element binding protein (CREB) concentrations ranged from 0.8 ng/ml to 1 ng/ml, whereas the proportions of phospho-CREB versus total CREB differed between the cell lines. Basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF) stimulated cAMP generation, whereas leukaemia inhibiting factor reduced some of their effects. Forskolin (0.05 and 1 mM) acted in synergy with FGF-2 and EGF; however, it caused pronounced donor to donor differences in the increase of cAMP and phospho-CREB levels. Additionally, dibutyryl-cAMP caused significant donor to donor variations in cell proliferation, possibly indicating a change of cell differentiation status. We speculate that similar donor diversity might be observed after cell stimulation with various G_s-protein-coupled receptor ligands. Heterogeneity of donor cell responses to stimulation of the cAMP pathway indicates the need for wide safety margins for S-MSC use in drug screening; nevertheless, knowledge of this heterogeneity might be useful for the design of donor-specific cell therapy.     

Cheiradone: a vascular endothelial cell growth factor receptor antagonist

Background  

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation) and in vivo (the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC₅₀) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated.     

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.     

The role of growth factors in maintenance of stemness in bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an active topic of research in regenerative medicine due to their ability to secrete a variety of growth factors and cytokines that promote healing of damaged tissues and organs. In addition, these secreted growth factors and cytokines have been shown to exert an autocrine effect by regulating MSC proliferation and differentiation. We found that expression of EGF, FGF-4 and HGF were down-regulated during serial passage of bone marrow-derived mesenchymal stem cells (BMSCs). Proliferation and differentiation potentials of BMSCs treated with these growth factors for 2 months were evaluated and compared to BMSCs treated with FGF-2, which increased proliferation of BMSCs. FGF-2 and -4 increased proliferation potentials at high levels, about 76- and 26-fold, respectively, for 2 months, while EGF and HGF increased proliferation of BMSCs by less than 2.8-fold. Interestingly, differentiation potential, especially adipogenesis, was maintained only by HGF treatment. Treatment with FGF-2 rapidly induced activation of AKT and later induced ERK activation. The basal level of phosphorylated ERK increased during serial passage of BMSCs treated with FGF-2. The expression of LC3-II, an autophagy marker, was gradually increased and the population of senescent cells was increased dramatically at passage 7 in non-treated controls. But FGF-2 and FGF-4 suppressed LC3-II expression and down-regulated senescent cells during long-term (i.e. 2 month) cultures. Taken together, depletion of growth factors during serial passage could induce autophagy, senescence and down-regulation of stemness (proliferation via FGF-2/-4 and differentiation via HGF) through suppression of AKT and ERK signaling.     

Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation

Neural stem/progenitor cell (NSPC) transplantation is a promising therapy for spinal cord injury (SCI). However, little is known about NSPC from the adult human spinal cord as a donor source. We demonstrate for the first time that multipotent and self-renewing NSPC can be cultured, passaged and transplanted from the adult human spinal cord of organ transplant donors. Adult human spinal cord NSPC require an adherent substrate for selection and expansion in EGF (epidermal growth factor) and FGF2 (fibroblast growth factor) enriched medium. NSPC as an adherent monolayer can be passaged for at least 9 months and form neurospheres when plated in suspension culture. In EGF/FGF2 culture, NSPC proliferate and primarily express nestin and Sox2, and low levels of markers for differentiating cells. Leukemia inhibitory factor (LIF) promotes NSPC proliferation and significantly enhances GFAP expression in hypoxia. In differentiating conditions in the presence of serum, these NSPC show multipotentiality, expressing markers of neurons, astrocytes, and oligodendrocytes. Dibutyryl cyclic AMP (dbcAMP) significantly enhances neuronal differentiation. We transplanted the multipotent NSPC into SCI rats and show that the xenografts survive, are post-mitotic, and retain the capacity to differentiate into neurons and glia.Together, these findings reveal that multipotent self-renewing NSPC cultured and passaged from adult human spinal cords of organ transplant donors, respond to exogenous factors that promote selective differentiation, and survive and differentiate after transplantation into the injured spinal cord.     

Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro

These experiments were aimed at using a recently developed serum-free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 X 10(5) RME cells per 60-mm type I collagen-coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F-12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater-than-tenfold increase in cell number over a 10-day culture period was obtained. Insulin could be replaced by physiological levels of insulin-like growth factor-I (IGF-I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3':5' monophosphate (cAMP) such as dibutyryl-cAMP (db-cAMP), prostaglandin E1 (PGE-1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17-B-estradiol (E2) to the I-, HC-, EGF-, and CT-containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.     

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.     

Antiproliferative effect of nitric oxide on epidermal growth factor-responsive human neuroblastoma cells

Addition of nitric oxide (NO) donors to NB69 neuroblastoma cells produced a cGMP-independent decrease in cell proliferation, without affecting cell viability or apoptosis. The potency of short half-life NO donors was higher when cell proliferation was stimulated by epidermal growth factor (EGF), as compared with cultures exposed to fetal calf serum (FCS). Immunoprecipitation and western blot analysis of the EGF receptor (EGFR) revealed a significant reduction of its EGF-induced tyrosine phosphorylation in cells treated with the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). When total cell lysates were subjected to western blotting, we observed that DEA-NO also reduced tyrosine phosphorylation in EGF-activated phosphoproteins, but not in those proteins whose tyrosine phosphorylation was evident in the absence of EGF. The effect of NO on EGFR transphosphorylation was concentration-dependent and transient, with a total recovery observed between 1.5 and 3 h after addition of DEA-NO to the cells. When cells were incubated for 15 min with DEA-NO and then washed, the EGFR transphosphorylation returned to control levels immediately, indicating that the interaction of NO with the receptor molecule was fully reversible. NB69 cells expressed both the neuronal and the inducible isoforms of NO synthase (NOS) when cultured in the presence of FCS; under this condition, the NOS inhibitor, N(omega)-nitro-L-arginine methyl ester, produced a small but significant increase in cell proliferation. The results suggest that NO is an endogenous antimitotic agent and that its interaction with EGFR contributes to cytostasis in NB69 cells.     

Epidermal growth factor can optimize a serum-free culture system for bone marrow stem cell proliferation in a miniature pig model

Bone marrow-derived mesenchymal stem cells have become an attractive cell source for periodontal ligament regeneration treatment because of their potential to engraft to several tissue types after injury. Most researchers have focused on the transplantation process, but few have paid attention to cell safety concerns and rapid proliferation before transplantation. Using serum-free medium to culture stem cells may be an effective method to avoid problems associated with exogenous serum and the addition of growth factors to promote cell proliferation. Here, we randomly divided our serum-free cultures and treated them with different levels of epidermal growth factor (EGF). We then evaluated changes in rates of cell adhesion, proliferation, apoptosis, and cell cycle ratio as well as their differentiation potential. The data showed that all of these parameters were significantly different when comparing serum-free cultures with and without 10 nM/L EGF (p?<?0.05/0.01); however, cells with 10 nM/L EGF did not respond differently than cells grown in standard serum-containing media without EGF (p?>?0.05). In summary, our results demonstrate that 10 nM/L EGF was the optimal dose for serum-free culture, which can replace traditional standard serum medium for in vitro expansion of miniature pig bone marrow-derived mesenchymal stem cells.     

Supplementation-dependent differences in the rates of embryonic stem cell self-renewal, differentiation, and apoptosis

Although it is known that leukemia inhibitory factor (LIF) supports the derivation and expansion of murine embryonic stem (ES) cells, it is unclear whether this is due to inhibitory effects of LIF on ES cell differentiation or stimulatory effects on ES cell survival and proliferation. Using an ES cell line transgenic for green fluorescent protein (GFP) expression under control of the Oct4 promoter, we were able to simultaneously track the responses of live Oct4-GFP-positive (ES) and -negative (differentiated) fractions to LIF, serum, and other growth factors. Our findings show that, in addition to inhibiting differentiation of undifferentiated cells, the administration of LIF resulted in a distinct dose-dependent survival and proliferation advantage, thus enabling the long-term propagation of undifferentiated cells. Competitive responses from the differentiated cell fraction could only be elicited upon addition of serum, fibroblast growth factor-4 (FGF-4), or insulin-like growth factor-1 (IGF-1). The growth factors did not induce additional differentiation of ES cells, but rather they significantly improved the proliferation of already differentiated cells. Our analyses show that, by adjusting culture conditions, including the type and amount of growth factors or cytokines present, the frequency of media exchange, and the presence or absence of serum, we could selectively and specifically alter the survival, proliferation, and differentiation dynamics of the two subpopulations, and thus effectively control population outputs. Our findings therefore have important applications in engineering stem cell culture systems to predictably generate desired stem cells or their derivatives for various regenerative therapies.     

A Global Transcriptome Analysis Reveals Molecular Hallmarks of Neural Stem Cell Death,Survival, and Differentiation in Response to Partial FGF-2 and EGF Deprivation

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

Background

Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood.In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO₂ conditions (ImF-H).

Results

During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions.

Conclusion

Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions.     

Differential properties of dentate gyrus and CA1 neural precursors

In the present article we investigated the properties of CA1 and dentate gyrus cell precursors in adult rodents both in vivo and in vitro. Cell proliferation in situ was investigated by rating the number of cells incorporating BrdU after kainate-induced seizures. CA1 precursors displayed a greater proliferation capacity than dentate gyrus precursors. The majority of BrdU-labeled cells in CA1 expressed Nestin and Mash-1, two markers of neural precursors. BrdU-positive cells in the dentate gyrus expressed Nestin, but only a few expressed Mash-1. In animals pretreated with the antimitotic azacytidine, the capacity of kainate to enhance the proliferation was higher in CA1 than in the dentate gyrus. Differences in intrinsic progenitor cell activity could underlie these different expansion capacities. Thus, we compared the renewal- expansion and multipotency of dentate gyrus and CA1 precursors isolated in vitro. We found that the dissected CA1 region, including the periventricular zone, is enriched in neurosphere-forming cells (presumed stem cells), which respond to either EGF or FGF-2. Dentate gyrus contains fewer neurosphere-forming cells and none that respond to FGF-2 alone. Neurospheres generated from CA1 were multipotent and produced neurons, astrocytes, and oligodendrocytes, while dentate gyrus neurospheres mostly produced glial cells. The analysis of the effects of EGF on organotypic cultures of hippocampal slices depicted similar features: BrdU and Nestin immunoreactivities increased after EGF treatment in CA1 but not in the dentate gyrus. These results suggest that CA1 precursors are more stem-cell-like than granule cell precursors, which may represent a more restricted precursor cell.     

Crosstalk of EGF-directed MAPK signalling pathways and its potential role on EGF-induced cell proliferation and COX-2 expression in human mesenchymal stem cells

Epidermal growth factor (EGF) promotes proliferation in human mesenchymal stem cells (hMSCs) during in vitro propagation. In this study, we investigated the effects of PI3K/AKT, ERK1/2, P38 and JNK on EGF signalling in hMSCs. The effects of EGF on MAPKs and PI3K/AKT crosstalk were investigated by immunoblotting; cyclooxygenase-2 (COX-2) expression was studied by real-time RT-PCR; and cell proliferation was evaluated by methylthiazolyl tetrazolium bromide assay. Our results showed that EGF immediately activated all four pathways, induced proliferation and increased COX-2 expression. Interestingly, inhibition of PI3K/AKT-enhanced EGF-stimulated ERK1/2 activity, and inhibition of ERK1/2 and JNK reduced AKT phosphorylation. Furthermore, EGF-induced proliferation as well as EGF-augmented COX2 expression was hindered by ERK1/2 and p38 inhibitors. The results of this study provide evidences to be used in extended proliferation of hMSCs for cell therapy.