Simple methods for cerebrospinal fluid collection in fetal, neonatal, and adult rat.

BACKGROUND: Cerebrospinal fluid (CSF) collection and its analysis are common medical practices useful in the diagnosis, therapy, and prevention of central nervous system (CNS) disorders. In recent years, several types of research have improved our insight into CSF and its role in health and disease. Yet, many characteristics of this fluid remain to be fully understood. NEW METHODS: Here, we describe how to collect CSF from embryonic, postnatal, and adult stages of the rat. In adults, CSF can be collected through simple stereotaxic surgery to expose the membrane overlying the cisterna magna (CM) of an anesthetized rat and collection of CSF through micropipette puncture through the membrane. In embryos and pups, CSF is aspirated, using a fire-polished micro-capillary pipette, from the CM of animals. RESULTS: Application of these methods provides the maximum volume of pure, uncontaminated CSF (embryonic day 19: 10-15 microliter, postnatal day 5: 20-30 microliter, adults: 100-200 microliter) with a success rate of approximately 95% in every age. COMPARISON WITH EXISTING METHODS: Compared to the existing protocols, these methods obtain considerable volumes of CSF, which may accelerate the measurement of biological markers in this fluid. Also, these techniques do not require surgical skills and according to the practical points mentioned during sampling, the procedures can be performed in rapid fashion. CONCLUSION: We describe simple methods for collecting CSF in live rats. These protocols provide clean, uncontaminated CSF for experiments to understand the exact role of this fluid in the development and maintenance of the CNS health.

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

The beneficial effects of chick embryo extract preconditioning on hair follicle stem cells: A promising strategy to generate Schwann cells.

The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells.

Melatonin protects against visible light-induced oxidative stress and promotes the implantation potential of mouse blastocyst in vitro.

Improvement of embryo culture media using antioxidant agents could help to improve embryo quality against environmental factors such as visible light and could overcome implantation failures. The usefulness of the melatonin against the effect of light on the expression of the primary implantation receptors, ErbB1 and ErbB4 on pre-implantation mouse embryo was investigated. Two-cell mouse embryos were exposed to the 1600 LUX light for 30 min then randomly divided into 3 groups including: Melatonin-Treated; Luzindole Treated and Simple media as a Control group. After 72-96  The expanded blastocysts were examined for morphological quality of the embryos by Hoechst and propidium iodide staining and for the expression of ErbB1 and ErbB4 by Real-time PCR and immunocytochemistry. The expression of the Sirt3 gene was also assayed. Furthermore, intracellular reactive oxygen species (ROS) levels and the total antioxidant capacity (TAC) were examined by DCFH-DA fluorescence intensity and radical cation respectively. The number of cells in the inner cell mass (ICM) and outer cell mass (OCM) were elevated significantly in the Melatonin-treated group suggesting increased viability and proliferation. Furthermore, we found that melatonin significantly increased the expression levels of ErbB1, ErbB4, and Sirt3 genes, and the protein expression of ErbB1, ErbB4 correlated with intracellular ROS levels and TAC significantly increased after melatonin treatment. Together, these results demonstrate that melatonin could be helpful to improve preimplantation embryos through its effects in decreasing ROS levels and increasing expression of implantation-related genes.

Folate Related Pathway Gene Analysis Reveals a Novel Metabolic Variant Associated with Alzheimer's Disease with a Change in Metabolic Profile.

Metabolic disorders may be important potential causative pathways to Alzheimer's disease (AD). Cerebrospinal fluid (CSF) decreasing output, raised intracranial pressure, and ventricular enlargement have all been linked to AD. Cerebral folate metabolism may be a key player since this is significantly affected by such changes in CSF, and genetic susceptibilities may exist in this pathway. In the current study, we aimed to identify whether any single nucleotide polymorphism (SNPs) affecting folate and the associated metabolic pathways were significantly associated with AD. We took a functional nutrigenomics approach to look for SNPs in genes for the linked folate, methylation, and biogenic amine neurotransmitter pathways. Changes in metabolism were found with the SNPs identified. An abnormal SNP in methylene tetrahydrofolate dehydrogenase 1 (MTHFD1) was significantly predictive of AD and associated with an increase in tissue glutathione. Individuals without these SNPs had normal levels of glutathione but significantly raised MTHFD1. Both changes would serve to decrease potentially neurotoxic levels of homocysteine. Seven additional genes were associated with Alzheimer's and five with normal ageing. MTHFD1 presents a strong prediction of susceptibility and disease among the SNPs associated with AD. Associated physiological changes present potential biomarkers for identifying at-risk individuals.

Knowledge of Neural Tube Defects and Prevention Through Folic Acid Use Among Women in Faisalabad, Punjab, Pakistan: A Cross-Sectional Survey.

Objective: Neural tube defects (NTDs) are one of the most common congenital abnormalities of the central nervous system and are associated with significant mortality, morbidity, and major life-long disability. Periconceptional folic acid reduces the risk of NTDs by up to 70%; however, in Pakistan, no public information program exists concerning the risks of NTDs or promoting folic acid use. As such, the aim of this study was to assess levels of knowledge about NTDs and folic acid use among women attending the gynaecology department of DHQ Hospital, Faisalabad, Pakistan. Design: A cross-sectional survey. Setting: The gynaecology department of the District Head Quarter (DHQ) Hospital in Faisalabad, Pakistan. Participants: Three hundred and fifty-five married women. Primary and Secondary Outcome Measures: Primary outcome measures included knowledge of NTDs and knowledge of folic acid use. Results: About 85.4% of participants had no knowledge of neural tube defects and 76.7% reported no knowledge about folic acid use. The majority of participants (86.2%) were unaware that folic acid protects against NTDs. Lack of knowledge of NTDs was significantly associated with education (P = 0.001), husband's education (P = 0.002), planned pregnancy (P = 0.002), sources of antenatal care (P = 0.003), knowledge of folic acid (P = 003), knowledge that folic acid protects against NTDs (P = 0.002), and health decision-making (P = 0.003). Knowledge of folic acid use was significantly associated with age (P = 0.000), education (P = 0.004), husband's education (P = 0.002), monthly income (P = 0.003), planned pregnancy (P = 0.003), pregnancy trimester (P < 0.001), sources of antenatal care (P < 0.001), knowledge of NTDs (P = 0.002), knowledge that folic acid protects against NTDs (P < 0.001), use of folic acid (P < 0.001), sources of vitamin information (P < 0.001), and health decision-making (P = 0.002). Conclusion: These findings highlight extremely low levels of knowledge about NTDs and folic acid use of women in Pakistan. There is an urgent need to increase knowledge and awareness of the risks of NTDs and preventative approaches, through health education programs delivered by trusted health professionals.

Intranasal application of stem cells and their derivatives as a new hope in the treatment of cerebral hypoxia/ischemia: a review.

Intranasal delivery of stem cells and conditioned medium to target the brain has attracted major interest in the field of regenerative medicine. In pre-clinical investigations during the last ten years, several research groups focused on this strategy to treat cerebral hypoxia/ischemia in neonates as well as adults. In this review, we discuss the curative potential of stem cells, stem cell derivatives, and their delivery route via intranasal application to the hypoxic/ischemic brain. After intranasal application, stem cells migrate from the nasal cavity to the injured area and exert therapeutic effects by reducing brain tissue loss, enhancing endogenous neurogenesis, and modulating cerebral inflammation that leads to functional improvements. However, application of this administration route for delivering stem cells and/or therapeutic substances to the damaged sites requires further optimization to translate the findings of animal experiments to clinical trials.

Experimental Models of SARS-CoV-2 Infection: Possible Platforms to Study COVID-19 Pathogenesis and Potential Treatments.

In December 2019, a novel coronavirus crossed species barriers to infect humans and was effectively transmitted from person to person, leading to a worldwide pandemic. Development of effective clinical interventions, including vaccines and antiviral drugs that could prevent or limit theburden or transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global health priority. It is thus of utmost importance to assess possible therapeutic strategies against SARS-CoV-2 using experimental models that recapitulate aspects of the human disease. Here, we review available models currently being developed and used to study SARS-CoV-2 infection and highlight their application to screen potential therapeutic approaches, including repurposed antiviral drugs and vaccines. Each identified model provides a valuable insight into SARS-CoV-2 cellular tropism, replication kinetics, and cell damage that could ultimately enhance understanding of SARS-CoV-2 pathogenesis and protective immunity.

The Beneficial Potential of Genetically Modified Stem Cells in the Treatment of Stroke: a Review.

The last two decades have witnessed a surge in investigations proposing stem cells as a promising strategy to treat stroke. Since growth factor release is considered as one of the most important aspects of cell-based therapy, stem cells over-expressing growth factors are hypothesized to yield higher levels of therapeutic efficiency. In pre-clinical studies of the last 15 years that were investigating the efficiency of stem cell therapy for stroke, a variety of stem cell types were genetically modified to over-express various factors. In this review we summarize the current knowledge on the therapeutic efficiency of stem cell-derived growth factors, encompassing techniques employed and time points to evaluate. In addition, we discuss several types of stem cells, including the recently developed model of epidermal neural crest stem cells, and genetically modified stem cells over-expressing specific factors, which could elevate the restorative potential of naive stem cells. The restorative potential is based on enhanced survival/differentiation potential of transplanted cells, apoptosis inhibition, infarct volume reduction, neovascularization or functional improvement. Since the majority of studies have focused on the short-term curative effects of genetically engineered stem cells, we emphasize the need to address their long-term impact.

The blood-CSF-brain route of neurological disease: The indirect pathway into the brain.

The brain is protected by the endothelial blood-brain barrier (BBB) that limits the access of micro-organisms, tumour cells, immune cells and autoantibodies to the parenchyma. However, the classic model of disease spread across a disrupted BBB does not explain the focal distribution of lesions seen in a variety of neurological diseases and why lesions are frequently adjacent to the cerebrospinal fluid (CSF) spaces. We have critically reviewed the possible role of a blood-CSF-brain route as a disease entry pathway into the brain parenchyma. The initial step of this pathway is the transfer of pathogens or immune components from the blood into the CSF at the choroid plexuses, where the blood-CSF barrier (BCSFB) is located. The flow of CSF results in disease dissemination throughout the CSF spaces. Access to the brain parenchyma from the CSF can then occur across the ependymal layer at the ventricular surface or across the pial-glial barrier of the subarachnoid space and the Virchow-Robin spaces. We have reviewed the anatomy and physiology of the blood-CSF-brain pathway and the brain barriers controlling this process. We then summarised the evidence supporting this brain entry route in a cross-section of neurological diseases including neuromyelitis optica, multiple sclerosis, neurosarcoidosis, neuropsychiatric lupus, cryptococcal infection and both solid and haematological tumours. This summary highlights the conditions that share the blood-CSF-brain pathway as a pathogenetic mechanism. These include the characteristic proximity of lesions to CSF, evidence of disruption of the brain barriers and the identification of significant pathology within the CSF. An improved understanding of pathological transfer through the CSF and across all brain barriers will inform on more effective and targeted treatments of primary and secondary diseases of the central nervous system.

Cerebral Folate Metabolism in Post-Mortem Alzheimer's Disease Tissues: A Small Cohort Study.

We investigated the cerebral folate system in post-mortem brains and matched cerebrospinal fluid (CSF) samples from subjects with definite Alzheimer's disease (AD) (n = 21) and neuropathologically normal brains (n = 21) using immunohistochemistry, Western blot and dot blot. In AD the CSF showed a significant decrease in 10-formyl tetrahydrofolate dehydrogenase (FDH), a critical folate binding protein and enzyme in the CSF, as well as in the main folate transporter, folate receptor alpha (FRα) and folate. In tissue, we found a switch in the pathway of folate supply to the cerebral cortex in AD compared to neurologically normal brains. FRα switched from entry through FDH-positive astrocytes in normal, to entry through glial fibrillary acidic protein (GFAP)-positive astrocytes in the AD cortex. Moreover, this switch correlated with an apparent change in metabolic direction to hypermethylation of neurons in AD. Our data suggest that the reduction in FDH in CSF prohibits FRα-folate entry via FDH-positive astrocytes and promotes entry through the GFAP pathway directly to neurons for hypermethylation. This data may explain some of the cognitive decline not attributable to the loss of neurons alone and presents a target for potential treatment.

Profound changes in cerebrospinal fluid proteome and metabolic profile are associated with congenital hydrocephalus.

The aetiology of congenital hydrocephalus (cHC) has yet to be resolved. cHC manifests late in rodent gestation, and by 18-22 weeks in human fetuses, coinciding with the start of the major phase of cerebral cortex development. Previously we found that cerebrospinal fluid (CSF) accumulation is associated with compositional changes, folate metabolic impairment and consequential arrest in cortical development. Here, we report a proteomics study on hydrocephalic and normal rat CSF using LC-MSMS and a metabolic pathway analysis to determine the major changes in metabolic and signalling pathways. Non-targeted analysis revealed a proteome transformation across embryonic days 17-20, with the largest changes between day 19 and 20. This provides evidence for a physiological shift in CSF composition and identifies some of the molecular mechanisms unleashed during the onset of cHC. Top molecular regulators that may control the shift in the CSF metabolic signature are also predicted, with potential key biomarkers proposed for early detection of these changes that might be used to develop targeted early therapies for this condition. This study confirms previous findings of a folate metabolic imbalance as well as providing more in depth metabolic analysis and understanding of cHC CSF.

The Implementation of Preconditioned Epidermal Neural Crest Stem Cells to Combat Ischemic Stroke. Comment on Othman, F.A.; Tan, S.C. Preconditioning Strategies to Enhance Neural Stem Cell-Based Therapy for Ischemic Stroke. Brain Sci. 2020, 10, 893.

In the recent review published in Brain Sciences, Othman and Tan suggested several preconditioning strategies to improve stem cell therapy after ischemic brain injury [...].

l-carnitine reduces the adverse effects of ROS and up-regulates the expression of implantation related genes in in vitro developed mouse embryos.

In vitro developed embryos are inevitably exposed to various reactive oxygen species (ROS) which may decrease the embryo's competence in assisted reproductive technology (ART) procedures. Optimization of embryo culture media using antioxidant agents could help to improve embryo quality and could overcome failures in current ART. The aim of this study was to evaluate the effects of l-carnitine (LC), an enhancer of mitochondrial activity and free radical scavenger, in culture media on early embryo competence and expression of ErbB1 and ErbB4 implantation related genes. Two-cell mouse embryos were cultured in the following four conditions: 1. LC group in media containing LC; 2.H 2O2 group exposed to H2O2 for 30 min and then transferred into a simple media; 3.H2O2+LC group exposed to H2O2 for 30 min and then transferred into a simple media containing LC; 4.the control group kept throughout in simple media. All groups were allowed to develop until the blastocyst stage. ErbB1 and ErbB4 expression were evaluated by Real-time PCR and immunocytochemistry. The expression of Sirt3 gene was also evaluated. Intracellular ROS levels were examined by DCFH-DA fluorescence intensity. In order to assess the morphological quality of the embryos, ICM and OCM number blastocyst cells were evaluated by using Hoechst and propidium iodide (PI) staining. ErbB1, ErbB4, ROS levels and cell number were compared across all in vitro groups. Our data reveal that LC significantly increases ErbB1 and ErbB4 gene and protein expression with intracellular ROS levels and Sirt3 gene expression significantly decreased after LC treatment. It is worth noting that an elevated cell number was observed in the LC-treated group compared with the other groups suggesting increased viability and/or proliferation. Our findings suggest that the use of LC could be helpful to improve preimplantation embryo culture media through its effects in decreasing ROS levels and the increase of implantation-related genes.

Embryonic cerebrospinal fluid formation and regulation.

The vertebrate brain is organized, from its embryonic origin and throughout adult life, around a dynamic and complex fluid, the cerebrospinal fluid (CSF). There is growing interest in the composition, dynamics and function of the CSF in brain development research. It has been demonstrated in higher vertebrates that CSF has key functions in delivering diffusible signals and nutrients to the developing brain, contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the patterning of the brain. It has also been shown that the composition and the homeostasis of CSF are tightly regulated following the closure of the anterior neuropore, just before the initiation of primary neurogenesis in the neural tissue surrounding brain cavities, before the formation of functional choroid plexus. In this review we draw together existing literature about the composition and formation of embryonic cerebrospinal fluid in birds and mammals, from the closure of the anterior neuropore to the formation of functional fetal choroid plexus, including mechanisms regulating its composition and homeostasis. The significance of CSF regulation within embryonic brain is also discussed from an evolutionary perspective.

The Effects of Embryonic Cerebrospinal Fluid on The Viability and Neuronal Differentiation of Adipose Tissue-Derived Stem Cells in Wistar Rats.

OBJECTIVE: The embryonic cerebrospinal fluid (e-CSF) contains various growth factors and morphogens. Recent studies showed that e-CSF plays significant roles in embryonic brain development. Adipose tissue-derived stem cells (ADSCs) have a mesodermal origin that can be differentiated into mesodermal and ectodermal lineages. This study aimed to evaluate the effects of e-CSF on the proliferation, viability, and neural differentiation of ADSCs in rats. MATERIALS AND METHODS: In this experimental study, adipose tissue was dissected out from the inguinal region of adult male rats. Then, ADSCs were isolated by enzymatic digestion from adipose tissues and mesenchymal cells were confirmed using the flow cytometry analysis that measured the cell surface markers including CD90, CD44, CD73, CD105, CD34, CD45, and CD11b. The multi-potential characteristics of ADSCs were assessed by osteogenic and adipogenic potentials of these cells. Under suitable in vitro conditions, ADSCs were cultured in DMEM supplemented with and without additional 10% e-CSF. These fluids were collected from Wistar rats at the E17, E18, and E19 gestational ages. Cellular proliferation and viability were determined using the MTT assay. Immunocytochemistry was used to study the expression of ß-III tubulin in ADSCs. The neurite outgrowth of cultured cells was assessed using the ImageJ software. RESULT: The results of the present study demonstrated that the viability of ADSCs in cell culture conditioned with E17 and E18 e-CSF were significantly increased in comparison with controls. Cultured cells treated with e-CSF from E18 and E19 established neuronal-like cells bearing long process, whereas no process was observed in the control groups or cultured cells treated with E17 e-CSF. CONCLUSION: This study showed that e-CSF has the ability to induce neuronal differentiation and viability in ADSCs. Our data support a significant role of e-CSF as a therapeutic strategy for the treatment of neurodegenerative diseases.

Subarachnoid cerebrospinal fluid is essential for normal development of the cerebral cortex.

The central nervous system develops around a fluid filled space which persists in the adult within the ventricles, spinal canal and around the outside of the brain and spinal cord. Ventricular fluid is known to act as a growth medium and stimulator of proliferation and differentiation to neural stem cells but the role of CSF in the subarachnoid space has not been fully investigated except for its role in the recently described "glymphatic" system. Fundamental changes occur in the control and coordination of CNS development upon completion of brain stem and spinal cord development and initiation of cortical development. These include changes in gene expression, changes in fluid and fluid source from neural tube fluid to cerebrospinal fluid (CSF), changes in fluid volume, composition and fluid flow pathway, with exit of high volume CSF into the subarachnoid space and the critical need for fluid drainage. We used a number of experimental approaches to test a predicted critical role for CSF in development of the cerebral cortex in rodents and humans. Data from fetuses affected by spina bifida and/or hydrocephalus are correlated with experimental evidence on proliferation and migration of cortical cells from the germinal epithelium in rodent neural tube defects, as well as embryonic brain slice experiments demonstrating a requirement for CSF to contact both ventricular and pial surfaces of the developing cortex for normal proliferation and migration. We discuss the possibility that complications with the fluid system are likely to underlie developmental disorders affecting the cerebral cortex as well as function and integrity of the cortex throughout life.

Altered folate binding protein expression and folate delivery are associated with congenital hydrocephalus in the hydrocephalic Texas rat.

Hydrocephalus (HC) is an imbalance in cerebrospinal fluid (CSF) secretion/absorption resulting in fluid accumulation within the brain with consequential pathophysiology. Our research has identified a unique cerebral folate system in which depletion of CSF 10-formyl-tetrahydrofolate-dehydrogenase (FDH) is associated with cortical progenitor cell-cycle arrest in hydrocephalic Texas (H-Tx) rats. We used tissue culture, immunohistochemistry, in-situ PCR and RT-PCR and found that the in-vitro proliferation of arachnoid cells is highly folate-dependent with exacerbated proliferation occurring in hydrocephalic CSF that has low FDH but high folate-receptor-alpha (FRα) and folate. Adding FDH to this CSF prevented aberrant proliferation indicating a regulatory function of FDH on CSF folate concentration. Arachnoid cells have no detectable mRNA for FRα or FDH, but FDH mRNA is found in the choroid plexus (CP) and CSF microvesicles. Co-localization of FDH, FRα and folate suggests important functions of FDH in cerebral folate transport, buffering and function. In conclusion, abnormal CSF levels of FDH, FRα and folate inhibit cortical cell proliferation but allow uncontrolled arachnoid cell division that should increase fluid absorption by increasing the arachnoid although this fails in the hydrocephalic brain. FDH appears to buffer available folate to control arachnoid proliferation and function.

In Vitro Effects of Wistar Rat Prenatal and Postnatal Cerebrospinal Fluid on Neural Differentiation and P roliferation of Mesenchymal Stromal Cells Derived from Bone Marrow.

OBJECTIVES: Cerebrospinal fluid (CSF) plays an important role in cortical development during the fetal stages. Embryonic CSF (E-CSF) consists of numerous neurotrophic and growth factors that regulate neurogenesis, differentiation, and proliferation. Mesenchymal stem cells (MSCs) are multi-potential stem cells that can differentiate into mesenchymal and non-mesenchymal cells, including neural cells. This study evaluates the prenatal and postnatal effects of CSF on proliferation and neural differentiation of bone marrow MSCs (BM-MSCs) at gestational ages E19, E20, and the first day after birth (P1). MATERIALS AND METHODS: In this experimental study, we confirmed the mesenchymal nature of BM-MSCs according to their adherence properties and surface markers (CD44, CD73 and CD45). The multi-potential characteristics of BMMSCs were verified by assessments of the osteogenic and adipogenic potentials of these cells. Under appropriate in vitro conditions, the BM-MSCs cultures were incubated with and without additional pre- and postnatal CSF. The MTT assay was used to quantify cellular proliferation and viability. Immunocytochemistry was used to study the expression of MAP-2 and ß-III tubulin in the BM-MSCs. We used ImageJ software to measure the length of the neurites in the cultured cells. RESULTS: BM-MSCs differentiated into neuronal cell types when exposed to basic fibroblast growth factor (b-FGF). Viability and proliferation of the BM-MSCs conditioned with E19, E20, and P1 CSF increased compared to the control group. We observed significantly elevated neural differentiation of the BM-MSCS cultured in the CSF-supplemented medium from E19 compared to cultures conditioned with E20 and P1 CSF group. CONCLUSIONS: The results have confirmed that E19, E20, and P1 CSF could induce proliferation and differentiation of BM-MSCs though they are age dependent factors. The presented data support a significant, conductive role of CSF components in neuronal survival, proliferation, and differentiation.