Exosome microRNAs in Amyotrophic Lateral Sclerosis: A Pilot Study.

The pathogenesis of amyotrophic lateral sclerosis (ALS), a lethal neurodegenerative disease, remains undisclosed. Mutations in ALS related genes have been identified, albeit the majority of cases are unmutated. Clinical pathology of ALS suggests a prion-like cell-to-cell diffusion of the disease possibly mediated by exosomes, small endocytic vesicles involved in the propagation of RNA molecules and proteins. In this pilot study, we focused on exosomal microRNAs (miRNAs), key regulators of many signaling pathways. We analyzed serum-derived exosomes from ALS patients in comparison with healthy donors. Exosomes were obtained by a commercial kit. Purification of miRNAs was performed using spin column chromatography and RNA was reverse transcribed into cDNA. All samples were run on the miRCURY LNATM Universal RT miRNA PCR Serum/Plasma Focus panel. An average of 29 miRNAs were detectable per sample. The supervised analysis did not identify any statistically significant difference among the groups indicating that none of the miRNA of our panel has a strong pathological role in ALS. However, selecting samples with the highest miRNA content, six biological processes shared across miRNAs through the intersection of the GO categories were identified. Our results, combined to those reported in the literature, indicated that further investigation is needed to elucidate the role of exosome-derived miRNA in ALS.

NMR Metabolomics for Stem Cell type discrimination.

Cell metabolism is a key determinant factor for the pluripotency and fate commitment of Stem Cells (SCs) during development, ageing, pathological onset and progression. We derived and cultured selected subpopulations of rodent fetal, postnatal, adult Neural SCs (NSCs) and postnatal glial progenitors, Olfactory Ensheathing Cells (OECs), respectively from the subventricular zone (SVZ) and the olfactory bulb (OB). Cell lysates were analyzed by proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy leading to metabolites identification and quantitation. Subsequent multivariate analysis of NMR data by Principal Component Analysis (PCA), and Partial Least Square Discriminant Analysis (PLS-DA) allowed data reduction and cluster analysis. This strategy ensures the definition of specific features in the metabolic content of phenotypically similar SCs sharing a common developmental origin. The metabolic fingerprints for selective metabolites or for the whole spectra demonstrated enhanced peculiarities among cell types. The key result of our work is a neat divergence between OECs and the remaining NSC cells. We also show that statistically significant differences for selective metabolites characterizes NSCs of different ages. Finally, the retrived metabolome in cell cultures correlates to the physiological SC features, thus allowing an integrated bioengineering approach for biologic fingerprints able to dissect the (neural) SC molecular specificities.

Adiponectin levels in the serum and cerebrospinal fluid of amyotrophic lateral sclerosis patients: possible influence on neuroinflammation?

BACKGROUND: Adiponectin (APN) is a key player in energy homeostasis strictly associated with cerebrovascular and neurodegenerative diseases. Since APN also belongs to anti-inflammatory-acting adipokines and may influence both neuroinflammation and neurodegenerative processes, we decided to study the APN levels in amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. METHODS: We assessed APN levels by ELISA immunoassay in both the serum and cerebrospinal fluid of a cohort of familial and sporadic ALS patients, characterized by normal body mass index and absence of dysautonomic symptoms. The screening of serum APN levels was also performed in patients affected by other neurological disorders, including fronto-temporal dementia (FTD) patients. Means were compared using the non-parametric Wilcoxon test, and Pearson's or Spearman's rho was used to assess correlations between variables. RESULTS: In the whole ALS group, serum APN levels were not different when compared to the age- and sex-matched control group (CTR), but a gender-specific analysis enlightened a significant opposite APN trend between ALS males, characterized by lower values (ALS 9.8 ± 5.2 vs. CTR 15 ± 9.7 µg/ml), and ALS females, showing higher amounts (ALS 26.5 ± 11.6 vs. CTR 14.6 ± 5.2 µg/ml). This sex-linked difference was significantly enhanced in familial ALS cases (p ≤ 0.01). The APN levels in ALS cerebrospinal fluids were unrelated to serum values and not linked to sex and/or familiarity of the disease. Finally, the screening of serum APN levels in patients affected by other neurological disorders revealed the highest serum values in FTD patients. CONCLUSIONS: Opposite serum APN levels are gender-related in ALS and altered in several neurological disorders, with the highest values in FTD, which shares with ALS several overlapping and neuropathological features. Further investigations are needed to clarify the possible involvement of APN in neuroinflammation and neurodegeneration. Possible involvement of APN in neuroinflammatory neurodegenerative diseases.

Phenotypic Modulation and Neuroprotective Effects of Olfactory Ensheathing Cells: a Promising Tool for Cell Therapy.

Olfactory Ensheathing Cells (OECs), exhibiting phenotypic characteristics of both astrocytes and Schwann Cells, show peculiar plasticity. In vitro, OECs promote axonal growth, while in vivo they promote remyelination of damaged axons. We decided to further investigate OEC potential for regeneration and functional recovery of the damaged Central Nervous System (CNS). To study OEC antigen modulation, OECs prepared from postnatal mouse olfactory bulbs were grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs) and analyzed for different neural specific markers. OEC functional characterizations were also achieved. Resistance of OECs to the neurotoxin 6-hydroxydopamine (6-OHDA) was analyzed by evaluating apoptosis and death. OEC neuroprotective properties were investigated by in vitro co-cultures or by addition of OEC conditioned medium to the neuroblastoma SH-SY5Y cells exposed to 6-OHDA. We observed: 1) modification of OEC morphology, reduced cell survival and marker expression in serum-free medium; 2) GF addition to serum-free medium condition influenced positively survival and restored basal marker expression; 3) no OEC apoptosis after a prolonged exposition to 6-OHDA; 4) a clear OEC neuroprotective tendency, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them potential clinical agents able to support injured CNS.

Dose dependent side effect of superparamagnetic iron oxide nanoparticle labeling on cell motility in two fetal stem cell populations.

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.

Labeling and tracking of human mesenchymal stem cells using near-infrared technology.

The recently developed near-infrared (NIR) light imaging technology combines low background noise with deep tissue penetration and readily allows imaging and tracking of NIR-labeled cells, following transplantation in small animal model of diseases. The real-time and longitudinal detection of grafted cells in vivo, as well as their rapid ex vivo localization, may further clarify graft interactions with the surrounding, in target and nontarget organs throughout the body, over time. The present chapter describes a protocol for (1) the efficient labeling of human mesenchymal stem cells (hMSCs) using a membrane intercalating dye, emitting in the NIR 815 nm spectrum; (2) the stereotaxic transplantation of NIR 815-hMSCs in rodent model of Parkinson's disease; and (3) the longitudinal in vivo detection of the grafted cells and the subsequent ex vivo imaging in selected tissues.

Biocompatible fluorescent nanoparticles for in vivo stem cell tracking.

Efficient application of stem cells to the treatment of neurodegenerative diseases requires safe cell tracking to follow stem cell fate over time in the host environment after transplantation. In this work, for the first time, fluorescent and biocompatible methyl methacrylate (MMA)-based nanoparticles (fluoNPs) were synthesized through a free-radical co-polymerization process with a fluorescent macromonomer obtained by linking Rhodamine B and hydroxyethyl methacrylate. We demonstrate that the fluoNPs produced by polymerization of MMA-Rhodamine complexes (1) were efficient for the labeling and tracking of multipotent human amniotic fluid cells (hAFCs); (2) did not alter the main biological features of hAFCs (such as viability, cell growth and metabolic activity); (3) enabled us to determine the longitudinal bio-distribution of hAFCs in different brain areas after graft in the brain ventricles of healthy mice by a direct fluorescence-based technique. The reliability of our approach was furthermore confirmed by magnetic resonance imaging analyses, carried out by incubating hAFCs with both superparamagnetic iron oxide nanoparticles and fluoNPs. Our data suggest that these finely tunable and biocompatible fluoNPs can be exploited for the longitudinal tracking of stem cells.

Vascular and parenchymal lesions along with enhanced neurogenesis characterize the brain of asymptomatic stroke-prone spontaneous hypertensive rats.

BACKGROUND AND OBJECTIVES: Spontaneously hypertensive stroke-prone rats (SHRSPs) develop hypertension, cerebrovascular abnormalities and a stroke phenotype in association with higher levels of proteinuria. Here, we focus on cerebral abnormalities preceding lesions detectable by MRI. METHODS: Longitudinal assessment of brain histology was performed in salt-loaded male SHRSPs (n = 26) and Wistar-Kyoto (WKY) normotensive control animals (n = 27). Groups of rats were sacrificed at different time points: Time 0, before the salt diet administration; Time 1, when proteinuria achieved 40 mg/day; Time 2, when proteinuria exceeded 100 mg/day. RESULTS: At Time 0, no brain lesions were observed. At Time 1, changes of the cortical penetrating arteries, vasogenic oedema, lacunae and focal cell loss appeared in SHRSPs and worsened at Time 2, although no lesions were yet detected by MRI. Staining for proliferation markers revealed a significant boost of cellular mitosis in the subventricular zone (SVZ) of SHRSPs. Moreover, we observed higher immunopositivity for nestin, glial fibrillary acidic protein and doublecortin (markers for neural stem cells, astrocytes and immature neurons, respectively). At Time 2, apoptotic caspase-3 as well as 4-hydroxynonenal-positive neurons were associated to decreased nestin and doublecortin staining. High expression levels of glial fibrillary acidic protein were maintained in the SVZ. No comparative alterations and SVZ activation were recorded in WKYs. CONCLUSION: Appearance of vascular changes in SHRSPs, before any MRI-detectable brain lesion, is coupled to active neural proliferation in the SVZ. With disease progression, only newborn astrocytes can survive, likely because of the neurotoxicity triggered by brain oedema and oxidative stress.

A reliable indirect cell-labelling protocol for optical imaging allows ex vivo visualisation of mesenchymal stem cells after transplantation.

We set out to assess the feasibility of exploiting expression of the mCherry gene, after lentiviral infection, in order visualise bone marrow-derived human mesenchymal stem cells (hMSCs) by optical imaging, and to provide proof of principle of this approach as a method for cell tracking and quantification in pre-clinical models. Commercial hMSCs were infected with a lentiviral vector carrying the mCherry gene under the control of the phosphoglycerate kinase promoter. After extensive in vitro culture, infected hMSCs were analysed for viability, morphology, differentiation capability, and maintenance of fluorescence. Thereafter, mCherry-positive cells were transplanted into unilaterally 6-hydroxy-dopamine lesioned rats (an experimental model of Parkinson's disease). Our analysis showed that hMSCs can be efficiently transduced with the lentiviral vector, retaining their biological features even in the long term. Intrastriatally transplanted mCherry-positive hMSCs can be detected ex vivo by a sensitive cooled CCD camera, both in the whole brain and in serial slices, and relatively quantified. Our protocol was found to be a reliable means of studying the viability of implanted hMSCs. mCherry labelling appears to be readily applicable in the post-transplantation tracking of stem cells and could favour the rapid development of new therapeutic targets for clinical treatments.

Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease.

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Neuroprotective effects of human mesenchymal stem cells on neural cultures exposed to 6-hydroxydopamine: implications for reparative therapy in Parkinson's disease.

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.

Metalloproteinase alterations in the bone marrow of ALS patients.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, nowadays considered as suitable candidate for autologous stem therapy with bone marrow (BM). A careful characterization of BM stem cell (SC) compartment is mandatory before its extensive application to clinic. Indeed, widespread systemic involvement has been recently advocated given that non-neuronal neighboring cells actively influence the pathological neuronal loss. We therefore investigated BM samples from 21 ALS patients and reported normal hematopoietic biological properties while an atypical behavior and impaired SC capabilities affected only the mesenchymal compartment. Moreover, by quantitative real-time approach, we observed altered Collagen IV and Metalloproteinase-9 levels in patients' derived mesenchymal stem cells (MSCs). Widespread metalloproteinase (MMPs) and their tissue inhibitor (TIMPs) alterations were established by multiplex ELISA analysis, demonstrating diffuse enzymatic variations in MSC compartment. Since MMPs act as fundamental effectors of extra-cellular matrix remodeling and stem cell mobilization, their modifications in ALS may influence reparative mechanisms effective in counteracting the pathology. In conclusion, ALS is further confirmed to be a systemic disease, not restricted to the nervous system, but affecting also the BM stromal compartment, even in sporadic cases. Therefore, therapeutic implantation of autologous BM derived SC in ALS patients needs to be carefully reevaluated.

Multiple neurogenic and neurorescue effects of human mesenchymal stem cell after transplantation in an experimental model of Parkinson's disease.

Stimulation of endogenous repair in neurodegenerative diseases, such as Parkinson's disease (PD), appears to be a novel and promising therapeutic application of stem cells (SCs). In fact SCs could propel local microenvironmental signals to sustain active endeavors for damaged neurons substitution, normally failing in non-supportive pathological surroundings. In this study, we demonstrated that two different doses of naïve human adult mesenchymal stem cells (hMSCs), implanted in the striatum of rats lesioned with 6-hydroxydopamine (6-OHDA), positively survived 23 days after transplantation. Their fate was directly influenced by the surrounding host environment while grafted hMSCs, dose dependently, regionally sustained the survival of striatal/nigral dopaminergic terminals and enhanced neurogenesis in the Subventricular Zone (SVZ). The number of proliferative cells (Ki67/Proliferating Cell Nuclear Antigen +) as well as neuroblasts migration significantly augmented in the lesioned striatum of transplanted animals compared to controls. No SVZ astrogenesis was detected in all experimental conditions, irrespectively of graft presence. Activation of endogenous stem cell compartments and rescue of dopaminergic neurons, supported by the persistent release of specific cytokine by MSCs in vivo, appeared in principle able to contrast the neurodegenerative processes induced by the 6-OHDA lesion. Our results suggest that reciprocal influences between grafted cells and endogenous neural precursors could be important for the observed neurorescue effect on several brain regions. Altogether, our data provide remarkable cues regarding the potential of hMSCs in promoting endogenous reparative mechanisms that may prove applicable and beneficial for PD treatment.

Amyotrophic lateral sclerosis: applications of stem cells - an update.

Neurodegenerative diseases are a growing public health challenge, and amyotrophic lateral sclerosis (ALS) remains a fatal incurable disease. The advent of stem cell therapy has opened new horizons for both researchers and ALS patients, desperately looking for a treatment. ALS must be considered a systemic disease affecting many cell phenotypes besides motor neurons, even outside the central nervous system. Cell replacement therapy needs to address the specific neurobiological issues of ALS to safely and efficiently reach clinical settings. Moreover, the enormous potential of induced pluripotent cells directly derived from patients for modeling and understanding the pathological mechanisms, in correlation with the discoveries of new genes and animal models, provides new opportunities that need to be integrated with previously described transplantation strategies. Finally, a careful evaluation of preclinical data in conjunction with wary patient choice in clinical trials needs to be established in order to generate meaningful results.

Transplantation of undifferentiated human mesenchymal stem cells protects against 6-hydroxydopamine neurotoxicity in the rat.

Stem cells have been increasingly recognized as a potential tool to replace or support cells damaged by the neurodegenerative process that underlies Parkinson's disease (PD). In this frame, human adult mesenchymal stem cells (hMSCs) have been proposed as an attractive alternative to heterologous embryonic or neural precursor cells. To address this issue, in this study we implanted undifferentiated hMSCs into the striatum of rats bearing a lesion of the nigrostriatal pathway induced by local injection of 6-hydroxydopamine (6-OHDA), a widely recognized rodent model of PD. Before grafting, cultured hMSCs expressed markers of both undifferentiated and committed neural cells, including nestin, GAP-43, NSE, beta-tubulin III, and MAP-2, as well as several cytokine mRNAs. No glial or specific neuronal markers were detected. Following transplantation, some hMSCs acquired a glial-like phenotype, as shown by immunoreactivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. More importantly, rats that received the striatal graft showed increased survival of both cell bodies and terminals of dopaminergic, nigrostriatal neurons, coupled with a reduction of the behavioral abnormalities (apomorphine-induced turning behavior) associated with the lesion. No differentiation of the MSCs toward a neuronal (dopaminergic) phenotype was observed in vivo. In conclusion, our results suggest that grafted hMSCs exert neuroprotective effects against nigrostriatal degeneration induced by 6-OHDA. The mechanisms underlying this effect remain to be clarified, although it is likely that the acquisition of a glial phenotype by grafted hMSCs may lead to the release of prosurvival cytokines within the lesioned striatum.

Stem cell transplantation in multiple sclerosis: safety and ethics.

Stem cell therapy is considered a promising strategy aiming at neuronal and glial cell replacement or neuroprotection in neurological diseases affecting the brain and spinal cord. Multiple Sclerosis (MS), characterized by inflammation-induced destruction of the myelin sheath surrounding axons leading to conduction deficits and variability of clinical signs, is not an exception. MS is considered an autoimmune disease and, in the last few years, an intense immunodepletion followed by autologous hematopoietic-stem-cell transplant (HSCT) is being assessed as potential therapeutical strategy for severe patients unresponsive to the immunomodulatory and immunosuppressive treatment. Partially supported by evidence in animal models and by anecdotal reports on the beneficial effects on MS patients with concomitant malignant diseases, HSCT programs for MS have been initiated worldwide and follow-up data are accumulating. A Consensus Meeting has been held in Milano (1998) providing a document that defined criteria for patient selection, transplantation procedures, and outcome evaluations. Nowadays the high number of patients already treated allows us to draw initial conclusions related to clinical efficacy. After careful monitoring of the available data and improvement of the procedure, safety seems not to be anymore an issue. Ethics of HSCT deserve, on the contrary, a profound evaluation: the procedure is a multistep process with manifold options, each step with different ethical implications. Even more difficult appears the definition of the MS patient selection criteria for HSCT. The informed consensus needs to be exhaustive for the full comprehension of a complex procedure. In conclusion, although HCST is today an established therapeutical option for MS patients, safety and ethical issues need to be further clarified.

Induction of neurotrophin expression via human adult mesenchymal stem cells: implication for cell therapy in neurodegenerative diseases.

In animal models of neurological disorders for cerebral ischemia, Parkinson's disease, and spinal cord lesions, transplantation of mesenchymal stem cells (MSCs) has been reported to improve functional outcome. Three mechanisms have been suggested for the effects of the MSCs: transdifferentiation of the grafted cells with replacement of degenerating neural cells, cell fusion, and neuroprotection of the dying cells. Here we demonstrate that a restricted number of cells with differentiated astroglial features can be obtained from human adult MSCs (hMSCs) both in vitro using different induction protocols and in vivo after transplantation into the developing mouse brain. We then examined the in vitro differentiation capacity of the hMSCs in coculture with slices of neonatal brain cortex. In this condition the hMSCs did not show any neuronal transdifferentiation but expressed neurotrophin low-affinity (NGFR(p75)) and high-affinity (trkC) receptors and released nerve growth factor (NGF) and neurotrophin-3 (NT-3). The same neurotrophin's expression was demonstrated 45 days after the intracerebral transplantation of hMSCs into nude mice with surviving astroglial cells. These data further confirm the limited capability of adult hMSC to differentiate into neurons whereas they differentiated in astroglial cells. Moreover, the secretion of neurotrophic factors combined with activation of the specific receptors of transplanted hMSCs demonstrated an alternative mechanism for neuroprotection of degenerating neurons. hMSCs are further defined in their transplantation potential for treating neurological disorders.

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential.

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.