Reelin regulates male mouse reproductive capacity via the sertoli cells.

Reelin plays important roles in brain development. Reeler mutant mice that lack the protein reelin (RELN) suffer from cell type- and region-dependent changes in their neocortical layers, and adult reeler mutant mice have dilated seminiferous tubules. Meanwhile, the mechanism by which Reelin regulates the spermatogenic cell development in mice and their reproductive abilities remains unclear. In the present study, we used reeler mutant mice to investigate the effects of Reelin on reproduction in mice. The results indicated variations in sex hormone expression among the reeler mice, indicating that they produce few offspring and their spermatogenic cells are irregularly developed. Moreover, glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha 1, Ras/extracellular regulated protein kinases (ERK), and promyelocytic leukemia zinc finger (PLZF)/chemokine (C-X-C motif) receptor 4 (CXCR4) serve as potential regulatory pathways that respond to the changes in sertoli cells and the niche of male germ cells. Our findings provided valuable insights into the role of reeler in the reproductive abilities of male mice and development of their spermatogonia stem cells.

H19 regulates the proliferation of bovine male germline stem cells via IGF-1 signaling pathway.

Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway.

Increased Neuronal Nitric Oxide Synthase in Alzheimer's Disease Mediates Spontaneous Calcium Signaling and Divergent Glutamatergic Calcium Responses.

Nitrosative stress is a feature of Alzheimer's disease (AD). Aims: We aimed to identify the cause underpinning increased nitric oxide (NO) in neurons and the impact of NO on neuronal function in AD. Results: We analyzed neuronal nitric oxide synthase (nNOS) protein levels in postmortem tissue and induced pluripotent stem cell (iPSC)-derived neurons from Alzheimer's patients and controls by immunohistochemistry and Western blots. Furthermore, we assessed the impact of modulating nNOS function or NO levels on neuronal glutamatergic signaling using calcium imaging. We show that nNOS protein levels are increased in early and severely affected brain regions of AD postmortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from late-onset Alzheimer's disease (LOAD) patients compared with controls, along with increased levels of nitrite, a stable marker of NO. Innovation: We observed a divergent functional impact of NO that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the AD neurons. Pharmacological scavenging of NO or inhibition of nNOS prevented aberrant spontaneous calcium signaling in AD neurons. Conclusion: Together these data identify increases in nNOS protein in AD. Functional data suggest that NO modulation of glutamatergic calcium signaling is neuroprotective under nonpathogenic conditions, with increased nNOS and NO contributing to dysregulated spontaneous calcium signaling in AD neurons.

Mesenchymal stem cell-secretome laden photopolymerizable hydrogels for wound healing.

Mesenchymal stem cell-derived secretome represents an emerging acellular therapeutic which possess significant opportunity for clinical applications due to its anti-inflammatory, immunomodulatory, and wound healing properties. However, maintaining therapeutic efficacy and ensuring stability of cell-based products is challenging, requiring a robust delivery method. Therefore, we designed a hydrogel-based scaffold loaded with CK Cell Technologies' proprietary Mesenchymal stem cell-secretome for controlled release treatment of acute and chronic wounds. We incorporated both conditioned media (CM) and extracellular vesicles (EVs) into gelatin methacryloyl (GelMA) hydrogels and demonstrated how we can tune the diffusive release of the EVs from them. To demonstrate viability of the approach, we developed a wound healing scratch assay where we see in situ release of CM and EVs promote enhanced migration of human dermal fibroblasts (hDFs). We see the colocalization of these EVs in the fibroblasts using fluorescent microscopy. Finally, as a surrogate for in vivo neovascularization, we conducted an in vitro tube formation assay for the MSC-secretome using matrigel-embedded human microvascular endothelial cells. By adding CM and EVs, we observe an increase in tubulogenesis. Collectively, our data demonstrates by tuning the GelMA properties, we can influence the controlled release of the MSC-secretome for a wound dressing and bandage application for chronic and acute wounds.

Induced pluripotent stem cells as tools for disease modelling and drug discovery in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that leads to a progressive decline in a person's memory and ability to communicate and carry out daily activities. The brain pathology in AD is characterized by extensive neuronal loss, particularly of cholinergic neurons, intracellular neurofibrillary tangles composed of the tau protein (NFTs) and extracellular deposition of plaques composed of ß-amyloid (Aß), a cleavage product of the amyloid precursor protein (APP). These two insoluble protein aggregates are accompanied by a chronic inflammatory response and extensive oxidative damage. Whereas dys-regulation of APP expression or processing appears to be important for the familial, early-onset form of AD, controversy exists between the "Baptists" (in favour of Aß) and the "Tauists" (in favour of tau) as to which of these two protein dysfunctions occur at the earliest stages or are the most important contributors to the disease process in sporadic AD. However, more and more "non-amyloid" and "non-tau" causes have been proposed, including, glycation, inflammation, oxidative stress and dys-regulation of the cell cycle. However, to get an insight into the ultimate cause of AD, and to prove that any drug target is valuable in AD, disease-relevant models giving insight into the pathogenic processes in AD are urgently needed. In the absence of a good animal model for sporadic AD, we propose in this review that induced pluripotent stem cells, derived from dermal fibroblasts of AD patients, and differentiated into cholinergic neurons, might be a promising novel tool for disease modelling and drug discovery for the sporadic form of AD.

Isolation and Characterisation of an Adipose-derived human mesenchymal stem cell line - 'CKC-Endeavour-1'.

Mesenchymal stem cells derived from adipose tissue (ADMSCs) are being increasingly considered in regenerative medicine-based clinical applications. Apart from possessing therapeutic applications themselves, ADMSCs also secrete a myriad of soluble factors which are promising candidates for treating several degenerative diseases such as osteoarthritis and neurodegenerative diseases, wound repair as well as for cosmeceutical purposes. In our research study, we successfully isolated ADMSCs in-house, now called CKC-Endeavour-1 from the lipoaspirate sample of a patient who underwent liposuction. The subsequent expansion of cells was performed in xeno-free and serum-free conditions and their characterisation was performed using tri-lineage differentiation studies. The levels of differentiation were assessed by staining and gene expression which was observed to be comparable between the in-house developed ADMSC cell line and the commercially purchased ADMSCs. Following characterisation, the secretory components from these MSCs, namely, conditioned media (ADMSC-CM) and exosomes (ADMSC-EXO) were harvested from CKC-Endeavour-1 under xeno-free, serum-free, and supplement-free conditions followed by lyophilisation in order to attempt to prolong its shelf-life. The comprehensive analysis of the secretome profile of ADMSC-CM using carried out using cytokine array and demonstrated the presence of 105 cytokines and growth factors. Also, clinical grade Izon columns were used to isolate the exosomes from ADMSC-CM obtaining exosomes in the size range of <200nm, analysed using nanoparticle tracking analysis. Overall, our study developed an ADMSC cell line, CKC-Endeavour-1, along with their CM and exosome (EXO) products under clinically safe conditions. Additionally, we have obtained a comprehensive understanding of the secreted factors present in the ADMSC-CM which could be further explored in detail to tap the best therapeutic benefits from them.

Neuroscience20 (BRAIN20, SPINE20, and MENTAL20) Health Initiative: A Global Consortium Addressing the Human and Economic Burden of Brain, Spine, and Mental Disorders Through Neurotech Innovations and Policies.

Neurological disorders significantly impact the world's economy due to their often chronic and life-threatening nature afflicting individuals which, in turn, creates a global disease burden. The Group of Twenty (G20) member nations, which represent the largest economies globally, should come together to formulate a plan on how to overcome this burden. The Neuroscience-20 (N20) initiative of the Society for Brain Mapping and Therapeutics (SBMT) is at the vanguard of this global collaboration to comprehensively raise awareness about brain, spine, and mental disorders worldwide. This paper aims to provide a comprehensive review of the various brain initiatives worldwide and highlight the need for cooperation and recommend ways to bring down costs associated with the discovery and treatment of neurological disorders. Our systematic search revealed that the cost of neurological and psychiatric disorders to the world economy by 2030 is roughly $16T. The cost to the economy of the United States is $1.5T annually and growing given the impact of COVID-19. We also discovered there is a shortfall of effective collaboration between nations and a lack of resources in developing countries. Current statistical analyses on the cost of neurological disorders to the world economy strongly suggest that there is a great need for investment in neurotechnology and innovation or fast-tracking therapeutics and diagnostics to curb these costs. During the current COVID-19 pandemic, SBMT, through this paper, intends to showcase the importance of worldwide collaborations to reduce the population's economic and health burden, specifically regarding neurological/brain, spine, and mental disorders.

Derivation of male germ cell-like lineage from human fetal bone marrow stem cells.

Mesenchymal stem cells derived from bone marrow are a well characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. Reports have shown that murine and human embryonic stem cells can differentiate into primordial germ cells and then to early gametes. Evidence has indicated that some adult stem cells also have the potential to differentiate into germ cells. Currently, there are no reports on directed differentiation of human mesenchymal stem cells into germ cells. This study investigated the ability of retinoic acid and testicular extracts to induce human bone marrow stem cells (hBMSC) to differentiate into male germ cells. It was found that a small population of hBMSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers OCT4, STELLA, NANOG and VASA, and male germ-ceil-specific markers such as DAZL, TH2, c-kit, beta(1)-integrin, ACR, PRMl, FSHR, STRA8 and SCP3, as analysed by reverse transcription-polymerase chain reaction and immunohistochemistry. These results demonstrated that hBMSC may differentiate into male germ cells and the same could be used as a potential source of cells for reproductive toxicological studies.

Recent advances in the derivation of germ cells from the embryonic stem cells.

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.

Derivation of a new human embryonic stem cell line, endeavour-1, and its clonal propagation.

Here we describe the derivation of a novel human embryonic stem (hES) cell line, Endeavour-1 (E1), its four new clonal lines (E1C1, E1C2, E1C3, E1C4), and their characterization. E1 and its clonal lines are propagated on human fetal fibroblasts (HFFs) derived and grown in a largely serum-free medium. Seven inner cell masses were isolated from 34 donated human embryos (27 survived), and one new hES cell line was obtained. E1 has been in culture for over 1 year and possesses all the typical features of stem cells, i.e., expression of stem cell surface markers (stage-specific embryonic antigens SSEA-3 and SSEA-4, and tumor recognition antigens TRA-1-60 and TRA-1-81), staining for alkaline phosphatase, and the presence of the pluripotent gene marker (nanog). This line shows pluripotency both under in vitro and in vivo conditions. E1 has a normal karyotype (46XX). Using our optimized procedure for cloning, four new clonal lines were derived from E1: E1C1, E1C2, E1C3, and E1C4. These clonal lines show normal characteristics: karyotype of that of the parent line (46XX) except for E1C3, which showed reciprocal translocation involving chromosomes 15 and 17; stem cell surface markers SSEA-4, TRA-1-60, and TRA-1-81; and gene expression for pluripotency (Nanog). All of these clonal lines formed embryoid bodies (EBs) in suspension cultures. After seeding, the EBs differentiated, forming cell lineages derived from all three germ layers as indicated by immunolocalization for the ectodermal marker beta-III tubulin, the mesodermal marker CD34, and the endodermal marker alpha-fetoprotein (AFP). There were subtle differences in the expression of these markers between clones. These clonal lines showed pluripotency in vivo. E1 and its clonal lines can differentiate to definitive endoderm after treatment with activin A, and, as indicated by expression of SOX17, FOXa2, and GATA-4 by RT-PCR, there are some subtle differences between these clonal lines. This may help in selecting clonal lines for specific lineage specification and for developing future cell therapy for various diseases.

Viral-free generation and characterization of a human induced pluripotent stem cell line from dermal fibroblasts.

Peripheral dermal fibroblasts (DF) from a healthy 56 year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage. Resource table.

Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells.

Diabetes mellitus affects a large number of men of reproductive age and it usually leads to serious reproductive disorders. However, the underlying mechanisms and specific therapies still remain largely unknown. We observed Leydig cell loss in the testes of diabetic mice. Continuous high glycemic status of testes stimulated expression of Caspase12, Grp78, and Chop, the three ERS response factors; this might induce cell cycle arrest and apoptosis of Leydig cells in response to ERS. In these diabetic mouse models, melatonin alleviated apoptosis of testicular stromal cell induced by ERS, and promoted SSCs self-renewal by recovering Leydig cells secretion of CSF1 after 8 weeks of treatment. To explore the relationship between CSF-1 and ERS in Leydig cells, we treated Leydig tumor cell line with an activator Tuniamycin and an inhibitor 4-Phenylbutyrate of ERS. Our data showed that the CSF-1 expression in mouse Leydig cell lines decreased six-fold while reversely increasing five-fold in the 4-Phenylbutyrate-treated group. Thus, melatonin likely alleviates the loss of Leydig cells in diabetic testes and provides a healthier niche for SSCs to self-renew and continually provide healthy sperm for male fertility.

Replicable Expansion and Differentiation of Neural Precursors from Adult Canine Skin.

Repopulation of brain circuits by neural precursors is a potential therapeutic strategy for neurodegenerative disorders; however, choice of cell is critical. Previously, we introduced a two-step culture system that generates a high yield of neural precursors from small samples of adult canine skin. Here, we probe their gene and protein expression profiles in comparison with dermal fibroblasts and brain-derived neural stem cells and characterize their neuronal potential. To date, we have produced >50 skin-derived neural precursor (SKN) lines. SKNs can be cultured in a highly replicable fashion and uniformly express a panel of identifying markers. Upon differentiation, they self-upregulate neural specification genes, generating neurons with basic electrophysiological functionality. This unique population of neural precursors, derived from mature skin, overcomes many of the practical issues that have limited clinical translation of alternative cell types. Easily accessible, neuronally committed, and patient specific, SKNs may have potential for the treatment of brain disorders.

Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization.

Here we describe the first report of three human embryonic stem cell (hESC) clones, hES 3.1, 3.2, and 3.3, derived from the parent line hES3 by sorting of single-cell preparations by flow cytometry. The viability of single-cell preparations before and after cell sorting remained >98%. The hESC were selected by size gating and forward-angle light scatter and were dispersed directly as single cell/ well into 96-well plates containing human fetal fibroblasts as feeder layers. Single stem cell dispersion into 96-well plates was confirmed by using cells from a hES3 line that constitutively expressed green fluorescence protein (eGFP) under similar conditions of flow cytometry. Three clones were obtained from the parent line hES3 -- hES3.1, 3.2, and 3.3 -- and they have been in continuous culture for more than 1 year. The cloning efficiency was less than <0.5%. These hESC clones show normal stem cell characteristics, such as undifferentiated growth, high nucleocytoplasmic ratio, the same karyotype as that of the parent line (46 XX), stem cell surface markers (i.e., SSEA3, SSEA4, OCT4, TRA-1-60, and TRA-1-81), and gene expression for pluripotency (Oct-4 and nanog). They all formed embryoid bodies in suspension cultures, and after seeding in culture plates they showed pluripotency in vitro by forming cell lineages derived from all three germ layers as indicated by expression of the ectodermal marker nestin, the mesodermal marker renin, and the endodermal markers alpha-fetoprotein and GATA6. All clones showed normal expression of alkaline phosphatase activity, a marker of in vitro pluripotency. When hESC clones (1-2 x 10(6) total) were injected into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice under the kidney capsule, all formed teratomas within 6-8 weeks. Analysis of the stem cell surface marker TRA-1-160 by flow cytometry showed nonsignificant (p < 0.05) differences between the clones and the parent line. The clones also differed in their expression of genes, with only one, hES 3.2, expressing the endodermal markers, i.e., alpha-fetoprotein and GATA6. The ability to produce clones from a parent hESC line rapidly by FACS sorting will help provide a homogeneous population of cells for achieving uniformed lineage specifications for future transplantation therapies and biomedical research.

Transgenic human fetal fibroblasts as feeder layer for human embryonic stem cell lineage selection.

Successful gene targeting in human embryonic stem (hES) cells requires the use of primary fibroblast feeder layers, which assist in the maintenance of the pluripotent state of hES cells. Such feeder layers must also survive any further selection strategy for hES cells. Here we report the production of a novel transgenic human fetal fibroblast (tHFF) as a feeder layer that is resistant to puromycin and can be used for gene targeting and selection of positive clones in hES cells. tHFFs survive under a wide range of puromycin concentrations (0.5-2 microg/ml) and also supports the undifferentiated growth of hES cells. We have demonstrated here that tHFFs are suitable for selecting Envy-hES cells that were transfected with a green fluorescent protein-small interfering RNA (GFP-siRNA) plasmid construct to induce GFP gene down-regulation. The later studies were designed to isolate and propagate stably knockdown cells. tHFFs thus can be used for targeting other genes that would serve as a model to select and understand the differentiation process in hES cells.

Differentiation of encapsulated embryonic stem cells after transplantation.

BACKGROUND: Embryonic stem cells (ESC) when transplanted into recipients with different major histocompatibility antigens may be rejected, especially as cells differentiate and expression of these antigens increases. One method to prevent rejection is to place the developing ESC in microcapsules. It is currently unknown what effect encapsulation has on the ability of ESC to differentiate. METHODS: Human ESC (hESC; hES03 line) and mouse ESC (mESC; R1 line) were encapsulated in 2.2% barium alginate and transplanted intraperitoneally in SCID and BALB/c mice respectively. Cell morphology, viability, and gene characterization were assessed after retrieving the capsules up to four weeks from SCID mice and three months from BALB/c mice. RESULTS: Encapsulation prevented hESC and mESC from forming teratomas up to four weeks and three months, respectively. mESC but not hESC formed aggregates within the capsules, which remained free of fibrosis. Some but not all the transplanted encapsulated hESC differentiated towards all three lineages, but more so towards an endodermal lineage as shown by increased expression of alpha fetoprotein. This was similar to what occurred when encapsulated and non-encapsulated hESC were cultured in vitro for two weeks. In contrast to the hESC, transplanted encapsulated mESC differentiated mostly towards an ectodermal lineage as shown by increased expression of nestin and glial fibrillary acidic protein. In vitro, encapsulated and nonencapsulated mESC also began to differentiate, but not down any specific lineage. CONCLUSIONS: Encapsulated ESC do differentiate, although along multiple pathways, both when transplanted and maintained in culture, just as nonencapsulated ESC do when removed from their feeder layer.

Derivation of Motor Neurons from three Clonal Human Embryonic Stem Cell Lines.

Human embryonic stem cells (hESC) demonstrate a remarkable proliferative and developmental potential and thus have huge therapeutic potential. To direct the differentiation of hESC to a specific lineage of high purity for cell transplantation is highly desirable. Here we describe a modified in vitro procedure to direct differentiation of three clonal hESC lines, hES 3.1, hES 3.2 and hES 3.3 efficiently to spinal motor neurons by using various differentiation factors namely retinoic acid (RA), sonic hedgehog (Shh), bone morphogenetic protein-2 (BMP-2) and Wnt3A. The highest number of motor neurons (58.0 +/- 7.6%) were obtained by an early treatment of embryoid bodies with a combination of RA + Shh from all the clonal hESC lines combined. The hES 3.1 line, however, produced relatively more motor neurons (69.5 +/- 11.8%) compared to other two hES clones, 3.2 (52.4 +/- 13.1%) and 3.3 (52.3 +/- 15.5%). Immunolocalisation studies revealed the expression of neuronal specific marker, beta omega-tubulin and motor neuron specific marker, HB9/HLXB9 in all the three hESC clones after 45 days of differentiation. The RT-PCR analyses showed the presence of the neuron-specific genes. This modified differentiation protocol provides a mean of obtaining an enriched population of motor neurons from hESC for possible use in studies of lineage development, drug discovery and also as a potential cell therapy for motor neuron disease.

Neuroprotective effects of apigenin against inflammation, neuronal excitability and apoptosis in an induced pluripotent stem cell model of Alzheimer's disease.

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative diseases, yet current therapeutic treatments are inadequate due to a complex disease pathogenesis. The plant polyphenol apigenin has been shown to have anti-inflammatory and neuroprotective properties in a number of cell and animal models; however a comprehensive assessment has not been performed in a human model of AD. Here we have used a human induced pluripotent stem cell (iPSC) model of familial and sporadic AD, in addition to healthy controls, to assess the neuroprotective activity of apigenin. The iPSC-derived AD neurons demonstrated a hyper-excitable calcium signalling phenotype, elevated levels of nitrite, increased cytotoxicity and apoptosis, reduced neurite length and increased susceptibility to inflammatory stress challenge from activated murine microglia, in comparison to control neurons. We identified that apigenin has potent anti-inflammatory properties with the ability to protect neurites and cell viability by promoting a global down-regulation of cytokine and nitric oxide (NO) release in inflammatory cells. In addition, we show that apigenin is able to protect iPSC-derived AD neurons via multiple means by reducing the frequency of spontaneous Ca(2+) signals and significantly reducing caspase-3/7 mediated apoptosis. These data demonstrate the broad neuroprotective action of apigenin against AD pathogenesis in a human disease model.

Neurogenesis and precursor cell differences in the dorsal and ventral adult canine hippocampus.

During evolution a unique anterior-posterior flexure posited the canine dentate gyrus in two distinct dorsal and ventral positions. We therefore sought to explore neurogenesis and neurogenic cell-related difference along the canine hippocampal dorsal-ventral axis. Post mortem histological analysis revealed 49.1% greater doublecortin (DCX)-positive cells and a 158.5% greater percentage of double labeled DCX-positive/neuronal nuclei (NeuN) positive cells in the dorsal subgranular zone compared to the ventral. We then show neural precursor cells isolated from fresh hippocampal tissue are capable of proliferating long term, and after differentiation, express neuronal and glial markers. Dorsal hippocampal isolates produced a 120.0% higher frequency of sphere-forming neural precursor cells compared to ventral hippocampal tissue. Histological DCX and neurosphere assay results were highly correlated. Overall, we provide the first evidence that the dorsal canine hippocampus has a markedly higher rate of adult neurogenesis than the ventral hippocampus, possibly related to a greater frequency of contributory neural precursor cells.

Alginate microcapsule as a 3D platform for the efficient differentiation of human embryonic stem cells to dopamine neurons.

Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction, cell proliferation and differentiation into specific lineages as well as tissue organization, it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers, particularly tyrosine hydroxylase (TH) by >100 folds after 2 weeks and at least 50% higher expression after 4 weeks respectively, compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH(+) cells co-expressed mdDA neuronal markers, forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4 weeks and secreted approximately 60pg/ml/10(6) cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages, particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies.