Ahmed Abd-Elmaksoud

Human Olfactory Bulb Neural Stem Cells expressing hNGF Restore Cognitive Deficit in Alzheimer's Disease Rat Model

In this study, we aim to demonstrate the fate of allogenic adult human olfactory bulb neural stem/progenitor cells (OBNSC/NPCs) transplanted into the rat hippocampus treated with ibotenic acid (IBO), a neurotoxicant specific to hippocampal cholinergic neurons that are lost in Alzheimers disease. We assessed their possible ability to survive, integrate, proliferate, and differentiate into different neuronal and glial elements: we also evaluate their possible therapeutic potential, and the mechanism(s) relevant to neuroprotection following their engraftment into the CNS milieu. OBNSC/NPCs were isolated from adult human olfactory bulb patients, genetically engineered to express GFP and human nerve growth factor (hNGF) by lentivirus‐mediated infection, and stereotaxically transplanted into the hippocampus of IBO‐treated animals and controls. Stereological analysis of engrafted OBNSCs eight weeks post transplantation revealed a 1.89 fold increase with respect to the initial cell population, indicating a marked ability for survival and proliferation. In addition, 54.71 ± 11.38%, 30.18 ± 6.00%, and 15.09 ± 5.38% of engrafted OBNSCs were identified by morphological criteria suggestive of mature neurons, oligodendrocytes and astrocytes respectively. Taken together, this work demonstrated that human OBNSCs expressing NGF ameliorate the cognitive deficiencies associated with IBO‐induced lesions in AD model rats, and the improvement can probably be attributed primarily to neuronal and glial cell replacement as well as the trophic influence exerted by the secreted NGF. J. Cell. Physiol. 230: 116–130, 2015.

Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis.

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.

Human olfactory bulb neural stem cells mitigate movement disorders in a rat model of Parkinson's disease

Parkinsons disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. Neural stem cells (NSCs) are multipotent stem cells that are capable of differentiating into different neuronal and glial elements. The production of DA neurons from NSCs could potentially alleviate behavioral deficits in Parkinsonian patients; timely intervention with NSCs might provide a therapeutic strategy for PD. We have isolated and generated highly enriched cultures of neural stem/progenitor cells from the human olfactory bulb (OB). If NSCs can be obtained from OB, it would alleviate ethical concerns associated with the use of embryonic tissue, and provide an easily accessible cell source that would preclude the need for invasive brain surgery. Following isolation and culture, olfactory bulb neural stem cells (OBNSCs) were genetically engineered to express hNGF and GFP. The hNFG‐GFP‐OBNSCs were transplanted into the striatum of 6‐hydroxydopamin (6‐OHDA) Parkinsonian rats. The grafted cells survived in the lesion environment for more than eight weeks after implantation with no tumor formation. The grafted cells differentiated in vivo into oligodendrocyte‐like (25 ± 2.88%), neuron‐like (52.63 ± 4.16%), and astrocyte ‐like (22.36 ± 1.56%) lineages, which we differentiated based on morphological and immunohistochemical criteria. Transplanted rats exhibited a significant partial correction in stepping and placing in non‐pharmacological behavioral tests, pole and rotarod tests. Taken together, our data encourage further investigations of the possible use of OBNSCs as a promising cell‐based therapeutic strategy for Parkinsons disease. J. Cell. Physiol. 230: 1614–1629, 2015.

Comparative expression of laminin and smooth muscle actin in the testis and epididymis of poultry and rabbit

The present investigation was conducted to demonstrate laminin and α smooth muscle actin (αSMA) in the testis and epididymis of adult chickens, Sudani ducks, pigeons, and rabbits. This study may represent the first indication for the presence of laminin in the male reproductive organs of birds and rabbits and might therefore serve as a milestone for further reports. In the testis of chicken, Sudani duck, pigeon, and rabbit, the laminin was localized in the basal lamina of the seminiferous tubules and of the peritubular myoid cells, in the testicular capsule and to a small extent in the vicinity of Leydig cells. The testicular vasculature also exhibited intense laminin immunostaining. Weak laminin staining was additionally seen in the cytoplasm of the duck Sertoli cells. In the epididymis, the basal lamina of the epididymal epithelium showed a distinctly positive reaction in all birds and rabbit. The basal lamina of the periductal myoid cells also showed a positive reaction. In the interductal tissue, laminin immunostaining was particularly observed in chicken, duck and pigeon. Laminin positive reaction was also seen in the epididymal vasculatures of all birds and rabbit. Interestingly, weak to moderate laminin staining was observed in the apical surface of the ciliated cells of the proximal and distal efferent ductules in chicken, duck and pigeon. αSMA positive reaction was seen in the testicular capsule and in the peritubular myoid cells of all birds and rabbit. In the testicular capsule, αSMA staining was either observed in the inner portion (chicken) or throughout the tunica albuginea (Sudani duck and pigeon), or in the outer aspect (rabbit). Distinct αSMA reaction was additionally observed in the testicular vasculature. In the epididymis of all birds and rabbit, the αSMA was particularly seen in the periductal and interductal myoid cells as well as in the epididymal vasculatures. No αSMA specific staining was however detected in the epididymal epithelium, fibrous lamina propria, and luminal spermatozoa of all birds and rabbits. In conclusion, the distribution of laminin and αSMA in the testis and epididymis might point out to their roles in the male reproduction.

Over-expression of hNGF in adult human olfactory bulb neural stem cells promotes cell growth and oligodendrocytic differentiation.

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.

In situ hybridization and immunohistochemical localization of leptin hormone and leptin receptor in the seminal vesicle and prostate gland of adult rat

The role of leptin in the regulation of male reproductive function is still a matter of debate. Knowledge about a possible source of leptin in the seminal plasma may therefore be helpful in identifying and elucidating the physiological role of leptin hormone in male reproduction. In our investigation, the expression of leptin and its long receptor isoform (Ob-Rb) was studied in adult male Wistar rats using RT-PCR, Southern blot, in situ hybridization and immunohistochemistry. RT-PCR analysis revealed the expression of both leptin and its Ob-Rb in the seminal vesicle and prostate gland. In situ hybridization also localized the mRNA transcripts of leptin and Ob-Rb in the glandular secretory epithelial cells of prostate gland and seminal vesicle. Immunohistochemistry detected the leptin hormone in the lining epithelium of both male genital glands. In conclusion, these findings suggest that the seminal vesicle and prostate gland could be the possible sources of leptin in the seminal plasma. This leptin might have a direct (paracrine, autocrine or both) effect on epithelial cells of the accessory male genital glands, on the spermatozoa via spermatozoan leptin receptors.

Expression and immunohistochemical localization of the neonatal Fc receptor (FcRn) in the mammary glands of the Egyptian water buffalo

Although a marginal placental transfer of maternal immunoglobulin (Ig) has been demonstrated in buffalo, the colostrum still provides the main source of immune components and nutrients to neonate buffalo calves. The neonatal Fc receptor (FcRn) transports maternal Ig across the gut wall and is involved in the transport of IgG in the mammary gland. In this study we used RT-PCR to examine the gene expression of FcRn in the mammary gland during several physiological states of the Egyptian water buffalo. The buffalo FcRn showed a high sequence homology to that of other mammalian species and especially the cow. Immunohistochemistry demonstrated positive immunolabelling of FcRn in the epithelial cells of the acini and ducts of the examined mammary gland tissue. Remarkable differences in both the cellular localization and in the intensity of FcRn immunopositivity were observed depending on the functional state of the mammary gland tissues. In late pregnancy, the FcRn immunolabelling was homogeneously distributed in the cytoplasm of the epithelial cells. In recently parturient animals, positive FcRn immunolabelling was mainly located at the luminal surface and apical cytoplasm of the mammary gland epithelium, while in dry and lactating animals, the FcRn immunolabelling was in the apical cytoplasm of the cells. The strongest FcRn immunolabelling was observed in late pregnancy and in recently parturient animals. In conclusion, the present data support the notion that FcRn might be involved in the transfer of maternal immunoglobulins and in the local defense mechanism of the mammary gland.

Localization of S-100 proteins in the testis and epididymis of poultry and rabbits.

The present investigation was conducted to demonstrate S-100 protein in the testis and epididymis of adult chickens, Sudani ducks, pigeons, and rabbits. This study may represent the first indication for the presence of S-100 in the male reproductive organs of these species and might therefore serve as a milestone for further reports. In the testis of chickens, pigeons and rabbits, intense S-100 was seen in Sertoli cells. S-100 was also seen in the endothelial lining of blood vessels in rabbit testis. On the contrary, no S-100 reaction was detected in the Sertoli cells of Sudani ducks. In epididymis, the localization of S-100 had varied according to species studied; it was seen in the basal cells (BC) of epididymal duct in duck, non-ciliated cells of the distal efferent ductules in pigeons and ciliated cells of the efferent ductules and BC of rabbit epididymis. Conversely, S-100 specific staining was not detected in the epithelial lining of the rooster and pigeon epididymal duct as well as the principal cells of the rabbit epididymis. In conclusion, the distribution of the S-100 proteins in the testis and epididymis might point out to its roles in the male reproduction.

Cholinergic and dopaminergic neuronal differentiation of human adipose tissue derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into various cell types such as cartilage, bone, and fat cells. Recent studies have shown that induction of MSCs in vitro by growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF2) causes them to differentiate into neural like cells. These cultures also express ChAT, a cholinergic marker; and TH, a dopaminergic marker for neural cells. To establish a protocol with maximum differentiation potential, we examined MSCs under three experimental culture conditions using neural induction media containing FGF2, EGF, BMP‐9, retinoic acid, and heparin. Adipose‐derived MSCs were extracted and expanded in vitro for 3 passages after reaching >80% confluency, for a total duration of 9 days. Cells were then characterized by flow cytometry for CD markers as CD44 positive and CD45 negative. MSCs were then treated with neural induction media and were characterized by morphological changes and Q‐PCR. Differentiated MSCs expressed markers for immature and mature neurons; β Tubulin III (TUBB3) and MAP2, respectively, showing the neural potential of these cells to differentiate into functional neurons. Improved protocols for MSCs induction will facilitate and ensure the reproducibility and standard production of MSCs for therapeutic applications in neurodegenerative diseases.

Morphological and glycohistochemical studies on the epididymal region of the Sudani duck (Cairina moschata).

In this study, the epididymal region of the Sudani duck was investigated using histological and lectin histochemical methods. Morphologically, the epididymal region of the Sudani duck is composed of extratesticular rete testis, proximal and distal efferent ductules, a short connecting duct, and epididymal ducts. Morphometric analysis of the epididymal region of Sudani duck revealed that the efferent ductules predominate in relation to the epididymal ducts. The distribution of sugar moieties within the epididymal region of the Sudani duck was investigated using ten different fluorescein isothiocyanate (FITC) conjugated lectins. In the rete testis epithelium, only PHA-L showed a positive reaction. Efferent ductules in contrary exhibited a wide range of lectin affinity whereas six positive lectins (Con A, LCA, PNA, WGA, PHA-L, PHA-E) were observed. In the connecting and epididymal ducts, four lectins (Con A, WGA, PHA-L, PHA-E) were also detected. GSA-I, UEA-I, and LTA were at all not evident in the epididymal region of the Sudani duck. In conclusion, the correlation between the large areas of the epididymal region occupied by the efferent ductules and the wide range of sugar affinity of this portion may confirm the speculation that efferent ductules might be the primary site of fluid reabsorption in the epididymal region of Sudani duck.