Abstract: Amyotrophic Lateral Sclerosis (ALS) is a devastating incurable disease. Stem-cell-based therapies represent a new possible strategy for ALS clinical research. The objectives of this Phase 1 clinical study were to assess the feasibility and toxicity of mesenchymal stem cell transplantation and to test the impact of a cell therapy in ALS patients. The trial was approved and monitored by the National Institute of Health and by the Ethics Committees of all participating Institutions. Autologous MSCs were isolated from bone marrow, expanded in vitro and analyzed according to GMP conditions. Expanded MSCs were suspended in the autologous cerebrospinal fluid (CSF) and directly transplanted into the spinal cord at a high thoracic level with a surgical procedure. Ten ALS patients were enrolled and regularly monitored before and after transplantation by clinical, psychological, neuroradiological and neurophysiological assessments. There was no immediate or delayed transplant-related toxicity. Clinical, laboratory, and radiographic evaluations of the patients showed no serious transplant-related adverse events. Magnetic resonance images (MRI) showed no structural changes (including tumor formation) in either the brain or the spinal cord. However the lack of post mortem material prevents any definitive conclusion about the vitality of the MSCs after transplantation. In conclusion, this study confirms that MSC transplantation into the spinal cord of ALS patients is safe and that MSCs might have a clinical use for future ALS cell based clinical trials.
Abstract: Amyotrophic lateral sclerosis (ALS) is a devastating incurable neurodegenerative disease that targets motor neurons, manifesting as a linear decline in muscular function and leading to death within 2 - 5 years of diagnosis. The vast majority of ALS cases are sporadic, the aetiopathology of which is incompletely understood. Recent data have implicated the microenvironment of the motor neuron as a primary target of the pathophysiology. Any experimental therapeutic approach to ALS is very difficult because of some peculiarities of the disease, such as the unknown origin, the spatial diffusion of motor neuron loss and the paucity of animal models. Despite such daunting challenges, in experimental models a number of potential benefits of stem cells in ALS therapy have been demonstrated: by providing non-compromised supporting cells such as astrocytes, microglia or growth factor-excreting cells, onset can be delayed and survival increased. Moreover, in animal models of acute or chronic motor neuron injury, neural stem cells implanted into the spinal cord have been shown to differentiate into motor neurons, with some evidence of axonal sprouting and formation of nerumuscular junctions with host muscle. Here we summarise and discuss current preclinical and clinical evidence regarding stem cells application in ALS, particularly focusing on methodological issues.
Abstract: BACKGROUND AIMS: Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro. METHODS: AF was harvested during routine pre-natal amniocentesis at 14-16 weeks of pregnancy. AF sample pellets were plated in alpha-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction. RESULTS: Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na(+) channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na(+) channels. CONCLUSIONS: These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.
Abstract: Amyotrophic Lateral Sclerosis is a progressive fatal neurodegenerative disease that targets motor neurons. Its origin is unknown but a main role of reactive astrogliosis and microglia activation in the pathogenesis has been recently demonstrated. Surrounding neurons with healthy adjoining cells completely stops motor neuron death in some cases. Hence stem cell transplantation might represent a promising therapeutic strategy. In this study MSCs were isolated from bone marrow of 9 patients with definite ALS. Growth kinetics, immunophenotype, telomere length and karyotype were evaluated during in vitro expansion. No significant differences between donors or patients were observed. The patients received intraspinal injections of autologous MSCs at the thoracic level and monitored for 4 years. No significant acute or late side effects were evidenced. No modification of the spinal cord volume or other signs of abnormal cell proliferation were observed. Four patients show a significant slowing down of the linear decline of the forced vital capacity and of the ALS-FRS score. Our results seem to demonstrate that MSCs represent a good chance for stem cell cell-based therapy in ALS and that intraspinal injection of MSCs is safe also in the long term. A new phase 1 study is carried out to verify these data in a larger number of patients.
Abstract: Amyotrophic lateral sclerosis (ALS) is a lethal disease affecting motoneurons. In familial ALS, patients bear mutations in the superoxide dismutase gene (SOD1). We transplanted human bone marrow mesenchymal stem cells (hMSCs) into the lumbar spinal cord of asymptomatic SOD1(G93A) mice, an experimental model of ALS. hMSCs were found in the spinal cord 10 weeks after, sometimes close to motoneurons and were rarely GFAP- or MAP2-positive. In females, where progression is slower than in males, astrogliosis and microglial activation were reduced and motoneuron counts with the optical fractionator were higher following transplantation. Motor tests (Rotarod, Paw Grip Endurance, neurological examination) were significantly improved in transplanted males. Therefore hMSCs are a good candidate for ALS cell therapy: they can survive and migrate after transplantation in the lumbar spinal cord, where they prevent astrogliosis and microglial activation and delay ALS-related decrease in the number of motoneurons, thus resulting in amelioration of the motor performance.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease lacking effective therapies. Cell replacement therapy has been suggested as a promising therapeutic approach for multiple neurodegenerative diseases, including motor neuron disease. We analyzed expanded mesenchymal stem cells (MSCs) isolated from sporadic ALS patients and compared them with MSCs isolated from healthy donors. MSCs were isolated from bone marrow by Percoll gradient and maintained in culture in MSC Medium until the third passage. Growth kinetics, immunophenotype, telomere length, and karyotype were evaluated during in vitro expansion. Osteogenic, adipogenic, chondrogenic, and neurogenic differentiation potential were also evaluated. No morphological differences were observed in the MSCs isolated from donors or patients. The cellular expansion potential of MSCs from donors and patients was slightly different. After three passages, the MSCs isolated from donors reached a cumulative population doubling higher than from patients but the difference was not statistically significant. No significant differences between donors or patients were observed in the immunophenotype analysis. No chromosomal alteration or evidence of cellular senescence was observed in any samples. Both donor and patient MSCs, after exposure to specific conditioning media, differentiated into adipocytes, osteoblasts, chondrocytes, and neuron-like cells. These results suggest that extensive in vitro expansion of patient MSCs does not involve any functional modification of the cells, including chromosomal alterations or cellular senescence. Hence, there is a good chance that MSCs might be used as a cell-based therapy for ALS patients.
Abstract: BACKGROUND AND AIMS: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. METHODS: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCl(4), respectively). Chimeric livers were analysed for expression of human transcripts and antigens. RESULTS: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for alpha-fetoprotein, CK18, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. CONCLUSIONS: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated.
Abstract: The enormous plasticity of mesenchymal stem cells (MSCs) suggests an improvement of a standard protocol of isolation and ex vivo expansion for experimental and clinical use. We isolated and expanded MSCs from bone marrow (BM) of pediatric and young adult donors, to analyze the growth kinetic, immunophenotype, telomere length, karyotype during ex vivo expansion. Seventeen BM samples were collected from young adult donors and 8 from pediatric donors. MSCs isolated from two groups showed no morphological differences while their cell growth was strictly related to the donor's age. The MSCs isolated from pediatric donors reached a cumulative PD almost twice as high as MSCs isolated from young adult donors after 112 days (10.2 +/- 1.9 versus 5.5 +/- 3.7). Furthermore, we analyzed the modulation of antigen expression in the MSCs isolated from two groups until 10th passage (77 days) and there was no significant difference between the modulation of antigen expression. In particular, at the first passage, MSCs showed a low contamination of hemopoietic cells which became insignificant in the following passages. There was a high expression of CD90, CD29, CD44 and CD105 and variable and moderate expression of CD166 and CD106 at the start of MSC culture and at each passage during expansion. No chromosomal alteration or evidence of cellular senescence were observed in all analyzed samples. All these data suggest that MSCs can be isolated and expanded from most healthy donors, providing for an autologous source of stem cells.
Abstract: OBJECTIVES: Our study was aimed to evaluate the feasibility and safety of intraspinal cord implantation of autologous mesenchymal stem cells (MSCs) in a few well-monitored amyotrophic lateral sclerosis (ALS) patients. METHODS: Seven patients affected by definite ALS were enrolled in the study and two patients were treated for compassionate use and monitored for at least 3 years. Bone marrow was collected from the posterior iliac crest according to the standard procedure and MSCs were expanded ex vivo according to Pittenger's protocol. The cells were suspended in 2 ml autologous cerebrospinal fluid and transplanted into the spinal cord by a micrometric pump injector. RESULTS: The in vitro expanded MSCs did not show any bacterial o fungal contamination, hemopoietic cell contamination, chromosomic alterations and early cellular senescence. No patient manifested major adverse events such as respiratory failure or death. Minor adverse events were intercostal pain irradiation and leg sensory dysesthesia, both reversible after a mean period of 6 weeks. No modification of the spinal cord volume or other signs of abnormal cell proliferation were observed. A significant slowing down of the linear decline of the forced vital capacity was evident in four patients 36 months after MSCs transplantation. CONCLUSIONS: Our results demonstrate that direct injection of autologous expanded MSCs into the spinal cord of ALS patients is safe, with no significant acute or late toxicity, and well tolerated. The clinical results seem to be encouraging.
Abstract: OBJECTIVE: Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. MATERIALS AND METHODS: We tested hMSCs in three previously reported experimental conditions made of alpha-modified minimum essential medium (alpha-MEM)/1 mM beta-mercaptoethanol (betaME), 10 microM alpha-MEM/retinoic acid (RA) or alpha-MEM/2% dimethylsulfoxide (DMSO) + 200 microM beta-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). RESULTS: hMSCs were isolated from bone marrow and expanded for several passages. In betaME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K+ currents related to two ether-Ã -go-go K+ channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K+ channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. CONCLUSION: In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.
Abstract: INTRODUCTION: Recently it has been shown in animal models of amyotrophic lateral sclerosis (ALS) that stem cells significantly slow the progression of the disease and prolong survival. We have evaluated the feasibility and safety of a method of intraspinal cord implantation of autologous mesenchymal stem cells (MSCs) in a few well-monitored patients with ALS. METHOD: Bone marrow collection was performed according to the standard procedure by aspiration from the posterior iliac crest. Ex vivo expansion of mesenchymal stem cells was induced according to Pittenger's protocol. The cells were suspended in 2 ml of autologous cerebrospinal fluid and transplanted into the spinal cord by a micrometric pump injector. RESULTS: No patient manifested major adverse events such as respiratory failure or death. Minor adverse events were intercostal pain irradiation (4 patients) which was reversible after a mean period of three days after surgery, and leg sensory dysesthesia (5 patients) which was reversible after a mean period of six weeks after surgery. No modification of the spinal cord volume or other signs of abnormal cell proliferation were observed. CONCLUSIONS: Our results appear to demonstrate that the procedures of ex vivo expansion of autologous mesenchymal stem cells and of transplantation into the spinal cord of humans are safe and well tolerated by ALS patients.
Abstract: Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combination of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34(+) placental blood cells were cultured in the presence of FL, TPO, KL +/- IL6 with SF medium, HS and FCS for up to 8 weeks. CD34(+), CFC, LTC-IC content was measured at intervals. To determine the in vivo repopulating capacity of expanded cells, CD34(+) expanded cells were transplanted in sublethally irradiated NOD/SCID mice. With the three growth factor combination the CD34(+) cell number increased steadily up to the 8 weeks of culture. CD34(+) cells were expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were considered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPO +/- IL6 it is possible to expand haemopoietic progenitor cells in a SF medium. Compared with serum-replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of whether this expansion might be useful in reducing post-transplant aplasia.
Abstract: The prolactin (PRL) receptor (R), a member of the cytokine hemopoietin receptor superfamily, has been shown to activate early differentiation steps along the erythroid pathway. In particular PRL, a product of bone marrow stroma, induces functional erythropoietin (EPO)-R on CD34+ hemopoietic progenitors. In this study, expression of EPO-R mRNA and responsiveness to EPO were assessed on enriched hemopoietic progenitor cells (HPC) from seven hyperprolactinemic and three normoprolactinemic patients and two normal subjects. Expression of EPO-R mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was found in HPC of four out of seven hyperprolactinemic patients but not in normoprolactinemic patients or normal donors. Development of EPO-dependent Colony Forming Unit-Erythroid (CFU-E) colonies in semi-solid medium was observed only in hyperprolactinemic patients (six out of seven). A much higher number of CFU-E colonies was observed in the four patients with a positive EPO-R message. We conclude from these data that abnormally high levels of PRL may increase the number of EPO-responsive hemopoietic precursors in vivo as they do in vitro. Since hyperprolactinemia associates in these patients with depressed EPO production, it may be regarded as a compensatory mechanism for the reduced availability of the hemopoietic factor.
Abstract: The stability of titanium dental implants is determined by osseointegration. Bone is a dynamic tissue continuously remodeled through resorption and formation, processes controlled by local cytokine production. This study investigated osseotropic cytokine expression in gingival mucosa, in the intraforamina and inferior first molar zones, during rehabilitation with implant-retained overdentures. Specimens were taken from six patients prior to placement of implants in the intraforamina bone; at connection of healing abutments; and 4, 8, and 12 months after prosthetic anchorage. Through semi-quantitative reverse-transcriptase polymerase chain-reaction, the following constitutively expressed cytokines were found at first surgical stage: interleukin-1, -6, and -8; small amounts of interleukin-11; stem cell factor; and transforming growth factor-beta1, -beta2, and -beta3. From the connection of healing abutments to 12 months after prosthetic anchorage, transforming growth factor-beta1, -beta2, and -beta3 were markedly higher than initial values. Expression of interleukin-6 and -8 decreased 8 months after prosthetic anchorage, while that of interleukin-1 increased at 12 months. In cultured gingival fibroblasts, modulation of cytokine secretion was also time-dependent. Cell culture supernatants influenced osteoclast-like multinucleated cell formation in long-term human marrow culture or osteoblast function, depending on the cytokine profile produced. These results are consistent with functional contributions of cytokines to osseointegration and minimization of posterior edentulous zone bone resorption.
Abstract: We report here that the progression of pancreatic carcinomas in tumor patients is associated with increased serum levels of both the soluble forms of CD95 ligand (CD95L/FasL) and its receptor, CD95 (Fas). Shedding of proteolytically processed soluble CD95L was also observed in pancreatic carcinoma cells in vitro, thus identifying one possible source of CD95L in patients' sera. Because the secreted forms of both CD95 and CD95L have been implicated previously in protection of cells from CD95-mediated cell death, we assessed the effect of soluble CD95L in supernatants of pancreatic carcinoma cells on viability of Jurkat T lymphocytes. We describe that (a) supernatants derived from cultured pancreatic carcinoma cells caused apoptosis of Jurkat cells; (b) soluble tumor-derived CD95L contributed significantly to this effect; and (c) in comparison to Jurkat cells, pancreatic carcinoma cells themselves revealed increased resistance to apoptosis induction by autocrine soluble CD95L. These results are consistent with the notion that in the microenvironment of pancreatic tumors, tumor-derived shed CD95L exerts paracrine pro-apoptotic effects. In addition, because it is released at high levels into the bloodstream, soluble CD95L may have systemic effects in tumor patients that reach beyond the microenvironment of the tumor site.
Abstract: In this study, we report coexpression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) in pancreatic carcinoma tissue associated with significantly elevated levels of both cytokines in the sera of pancreatic carcinoma patients. Using conditioned media (CM) of pancreatic carcinoma cells, we further demonstrate that tumor cell-derived TGF-beta and IL-10 inhibited in an additive fashion both proliferation and the development of Th1-like responses in peripheral blood mononuclear cell (PBMC) preparations derived from normal donors. The antiproliferative and Th1-suppressive activities contained in CM of pancreatic carcinoma cells were due primarily to IL-10 and/or TGF-beta, as shown by the capacity of cytokine-specific neutralizing antibodies to reverse these effects. Finally, as compared to normal controls, PBMC derived from pancreatic carcinoma patients displayed a Th2-like cytokine expression pattern upon activation with either anti-CD3 antibody or Staphylococcus aureus strain Cowan I. Taken together, these results suggest that aberrant production of TGF-beta and IL-10 in pancreatic tumor patients skews T-cell cytokine production patterns in favor of a Th2 immunophenotype.
Abstract: Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an anti-human PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.
