Fetal Nervous Tissue Research Articles

Abstract 3778: Uncovering the role of glioma stem cells in glioblastoma chemo-resistance and recurrence

Abstract Glioblastoma (GBM) is the most common malignant brain tumor. Despite multimodal aggressive therapies, the median survival after diagnosis is 14 months. The failure of current therapies is due to the presence within the tumor of a subpopulation of cancer cells with stem-like properties, called glioma stem cells (GSCs), which are responsible for chemo-resistance and recurrence. The molecular mechanisms involved in chemo-resistance of this cell subpopulation and its role in recurrence are still largely unknown. In this work, we isolated GSCs from seven recurrent GBM patients and we performed single-cell RNA-sequencing (scRNA-seq) on them. The analysis showed the persistence of a conserved neurodevelopmental hierarchy in recurrent GBM with a glial-like progenitor population at the top. Furthermore, we extended this analysis by sequencing the tumor bulk of seven recurrent GBMs in order to investigate deeply cancer heterogeneity in recurrences. Our data displayed the presence of many different cell populations that recapitulate the multiple steps of the physiological neural differentiation, suggesting that this capability is not affected by standard therapies. Afterwards, we confirmed the stem-like properties of our recurrent GSCs, verifying by immunofluorescence the expression of several stem-ness markers, such as PROM1, MASH1, OLIG2 and SOX2, and assessing the tumorigenicity in vivo of these cells. Standard therapies are able to eliminate only differentiated cancer cells, but they do not eradicate the cancer stem cell subpopulation. In order to defeat GBM and avoid recurrence, GSCs have to become the goal of future therapies. Considering chemo-resistance as an intrinsic and natural property of these cells, we performed a pathways analysis comparing the scRNA-seq datasets of recurrent GBMs with scRNA-seq datasets of primary GBMs and fetal nervous tissues to identify shared molecular pathways. Intriguingly, several pathways involved in DNA repair, cell cycle control and stem cell maintenance, such as BRCA1, ATM, E2F4 and FOXM1 pathways, were uniformly overexpressed by the progenitor cells of the three groups, representing new molecular targets for therapeutic purposes. Afterwards, we tested in vitro an E2F4 inhibitor on GSCs derived from de novo and recurrent GBMs, obtaining stunning results in terms of cell growth inhibition and cell death induction. Furthermore, we pretreated recurrent GCSs with this molecule and we injected them in mice: data showed the ability of the drug to reduce tumor growth sharply and prolong the survival. Currently, we are evaluating the efficacy in vivo of this compound on a recurrent GBM mouse model, administering the drug with intracerebroventricular pumps. This project could seriously help to decipher GSCs chemo-resistance, as well as their role in GBM relapse, also providing the necessary knowledge for the development of new targeted therapies. Citation Format: Gabriele Riva, Charles Couturier, Phuong Uyen Le, Xiaohua Yan, Yu Chang Wang, Marie Christine Guiot, Ioannis Ragoussis, Kevin Petrecca. Uncovering the role of glioma stem cells in glioblastoma chemo-resistance and recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3778.

Adult Neural Stem Cells: Basic Research and Production Strategies for Neurorestorative Therapy.

Over many decades, constructing genetically and phenotypically stable lines of neural stem cells (NSC) for clinical purposes with the aim of restoring irreversibly lost functions of nervous tissue has been one of the major goals for multiple research groups. The unique ability of stem cells to maintain their own pluripotent state even in the adult body has made them into the choice object of study. With the development of the technology for induced pluripotent stem cells (iPSCs) and direct transdifferentiation of somatic cells into the desired cell type, the initial research approaches based on the use of allogeneic NSCs from embryonic or fetal nervous tissue are gradually becoming a thing of the past. This review deals with basic molecular mechanisms for maintaining the pluripotent state of embryonic/induced stem and reprogrammed somatic cells, as well as with currently existing reprogramming strategies. The focus is on performing direct reprogramming while bypassing the stage of iPSCs which is known for genetic instability and an increased risk of tumorigenesis. A detailed description of various protocols for obtaining reprogrammed neural cells used in the therapy of the nervous system pathology is also provided.

Дослідження дегенеративних процесів у норадреналінергічних системах головного мозку щурів з тяжкою черепно-мозковою травмою та вплив на них трансплантації фетальної нервової тканини

Проведено дослідження вмісту норадреналіну та рівня експресії гена Вах у корі лівої та правої півкуль, гіпоталамусі, гіпокампі та середньому мозку щурів через 30 днів після експериментальної тяжкої черепно-мозкової травми та трансплантації фетальної нервової тканини. Встановлено, що тяжка черепно-мозкова травма призводить до зниження вмісту норадреналіну в корі травмованої півкулі та гіпоталамусі, а також до підвищення рівня проапоптичного гена Вах у всіх досліджених структурах головного мозку, що свідчить про наявність деструктивних процесів у мозку тварин у віддалений період після травми. Трансплантація фетальної нервової тканини експериментальним щурам з тяжкою черепно-мозковою травмою сприяла нормалізації досліджуваних показників.

Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray

ObjectsOur objective was to develop an oligonucleotide DNA microarray (OMA) for genome-wide microRNA profiling and use this method to find miRNAs, which control organic development especially for nervous system.Materials and methodsEighteen organic samples included cerebrum and spinal cord samples from two aborted human fetuses. One was 12 gestational weeks old (G12w) and the other was 24 gestational weeks old (G24w). Global miRNA expression patterns of different organs were investigated using OMA and Northern blot.ConclusionThe OMA revealed that 72–83% of miRNAs were expressed in human fetal organs. A series of microRNAs were found specifically and higher-expressed in the human fetal nervous system and confirmed consistently by Northern blot, which may play a critical role in nervous system development.

An improved method for the expression of TRH in serum-supplemented primary cultures of fetal hypothalamic cells

Primary cultures of dispersed cells from fetal nervous tissue are extensively used for studying multiple neuronal properties. Analyses of the developmental expression of thyrotropin releasing hormone (TRH; pglu-his-pro-NH 2) biosynthesis in primary cultures of fetal dissociated hypothalamic cells have shown that cellular TRH levels per dish increase with time in culture, after a lag period of a few days, but do not attain the values observed in vivo, hampering its use as a model system for the study of peptide biosynthesis and release. We have demonstrated that homologous conditioned medium (CM) enhances TRH expression in dissociated cell cultures from fetal mice hypothalamus, maintained in presence of serum. We report here experimental conditions that allow the expression, during the second or third week in vitro, of higher cellular TRH levels than previously described in primary cultures of dissociated hypothalamic cells from 17-day rat fetuses. The medium used was Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%), vitamins, glucose, glutamine and insulin (DMEM-S). Cellular levels of TRH/mg protein increased with cell density between 1 and 2.7×10 6 cells per 35-mm dish. Addition of 10 −5 M cytosine arabinoside (CAr) at the 4th day in vitro (DIV) improved TRH cell content per dish compared to addition at 5 DIV; 2.5–5×10 −5 M bromodeoxyuridine added at seeding reduced cell survival and did not enhance TRH levels, in comparison to CAr-treated cultures. Addition of ascorbic acid (0.5–1×10 −4 M) increased TRH levels per dish. Substitution of DMEM by DMEM-F12 (1:1) did not improve TRH levels. Cellular levels of TRH, in Neurobasal plus B27 (a serum-free medium), were similar to levels in serum-supplemented media. In the optimized conditions, a small number of pro-TRH mRNA expressing cells (2% of total cells) was detected by in situ hybridization; 40% coexpressed the pro-protein convertase PC1 mRNA. Conditioning the medium, controlling glial proliferation, and adding ascorbic acid improved the expression of TRH in primary culture of hypothalamic cells in DMEM-S.

Survival of neurons and interstitial cells of Cajal after autotransplantation of myenteric ganglia from small intestine in the lethal spotted mouse.

To avoid mutilating surgery in the treatment of distal aganglionosis, transplantation of autologous nervous elements to the affected intestine would be an attractive option. This treatment modality has emerged as a possible alternative for different brain disorders, mostly using fetal nervous tissue. Our objective was to evaluate whether myenteric ganglia (MG) and interstitial cells of Cajal (ICC) could survive a transplantation procedure and to evaluate possible differences between animals with distal colonic aganglionosis (lethal spotted mice) and their healthy littermates. Autologous transplantation of MG with adherent smooth muscle from small intestine to the subcapsular space of the kidney was performed in mice 3-12 weeks of age. The transplants were evaluated 5 to 9 days postoperatively. The presence of myenteric neurons in the transplants was registered using immunohistochemical detection of different neurotransmitters and markers. For identification of ICC antibodies against c-kit, a cell surface tyrosine-kinase receptor, were used. The transplants showed overall good survival. Neurons containing the general neuronal marker protein gene-related product, the neuronal nitric oxide synthesizing enzyme, and the neuropeptides vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, calcitonin gene-related peptide, galanin, substance P, and neuropeptide Y could be shown throughout the transplants. ICC were consistently seen in the grafted tissue among the smooth muscle cells, particularly in the deep muscular plexus, and within the MG. No obvious differences in ICC or enteric neuronal tissue survival, or in the frequency of the various neuronal populations displayed could be detected between the two groups of animals. These findings support the use of autologous MG for further research on transplantation of enteric ganglia as a possible alternative treatment for colonic aganglionosis.

Threshold of seizure activity of the brain and higher nervous activity in the postresuscitation period for intracerebral allotransplantation of fetal neocortex tissue

The threshold of seizure activity of the brain, long-term memory, and learning ability are studied in Wistar rats for bilateral transplantation of fetal nervous tissue in area CA1 of the hippocampus. The grafts are performed on the 2nd, 7th, 14th and 30th days after clinical death caused by asphyxia. A neurotransplantation performed on the 2nd day of the postresuscitation period is found to prevent seizure activity, whereas that performed on the 7th–14th days results in a sharp decline or cessation of spontaneous and induced epileptiform convulsive seizures, prolonged preservation of the long-term memory trace, an improvement of learning ability, and a lessening of defensive and phobic behavior in a large proportion of the animals.

Opioid peptides improve human fetal nervous tissue survival after cryopreservation in culture

The study was carried out on the 18–20-week human fetal brain by culturing organotypic and dissociated cells of the cerebral cortex and the corpora quadrigemina area. Tissue viability was assessed from the formation of growth cones and neuroglial bundles, as well as of glial cell formation. The study showed that after tissue freezing in organotypic cultures, viable cells were detected in not more than 10% of grafts. The addition of opioid peptides noticeably (by 1.3–2 times) increased the number of surviving cells. This regularity was not observed in dissociated cultures.

Amelioration of memory deficits by intraperitoneal injections of substance P in rats with hippocampal lesions and grafts of fetal nervous tissue

Amelioration of memory deficits by intraperitoneal injections of substance P in rats with hippocampal lesions and grafts of fetal nervous tissue

Human fetal tissues grafted to rodent hosts: structural and functional observations of brain, adrenal and heart tissues in oculo.

The potential for growth and development of human tissue grafts was explored by transplantation to the anterior chamber of the eye of rats and mice. Tissues were obtained from therapeutic abortions, performed in the eighth to twelfth week of gestation, using a slight modification of routine vacuum aspirations. Recipients were either adult rats immunosuppressed with cyclosporin A and protected with antibiotics, or nude immunodeficient Balb C mice. Catecholamine-rich tissues such as chromaffin cells from the adrenal medulla, sympathetic ganglia, central dopamine neuroblasts from the substantia nigra, and noradrenaline neuroblasts from the locus coeruleus all survived grafting, and in many cases formed nerve fibers that invaded the host iris. Similarly, central serotonin neurons from developing raphe nuclei grafts were able to innervate host irides. Human fetal cerebellar and cerebral cortical transplants continued their development in rat host eyes. Extracellular recordings from such cerebellar and cortical grafts revealed spontaneously active cells with immature action potential waveforms. Spinal cord grafts also survived and contained substance P-immunoreactive neurons. Dorsal root ganglia were able to form nerve fibers invading the host iris, as evidenced by neurofilament immunohistochemistry. Heart tissue survived and manifested spontaneous rhythmic contractions in oculo. Both human cortex cerebri and heart tissue grafts became innervated by sympathetic adrenergic nerve fibers from the rat host iris. Thus both graft-to-host and host-to-graft neuronal connections may be established between man and rat. Taken together, these data suggest that transplantation of human fetal nervous tissues to the anterior chamber of immunosuppressed or immunodeficient rodent hosts yields a unique model system for studies of human brain development, developmental disturbances, connectivity, and the action of drugs.

Effects of fetal thyroid states on RNA, DNA, and tubulin content in neonatal rat brain.

3,5-Dimethyl-3'-isopropyl-L-thyronine (DIMIT)-induced fetal hyperthyroid rats showed a marked accumulation of intracellular tubulin content in the cerebral cortex, hypothalamus, and cerebellum at 1 day old. When the fetuses were transplacentally radio-thyroidectomized with the administration of [131I]-Na, the brain weight was not changed at 1 day old. The DNA content was not affected by the radio-thyroidectomy (Tx), but intracellular RNA concentration (per DNA) was increased in the hypothalamus and cerebellum at that age. The DIMIT-supplement to Tx-fetuses failed to restore these abnormal values to normal. Delayed effects of the fetal Tx were observed in 11-day-old infants. These neonates showed decreased weight gain in the body, brain, cerebral cortex, and cerebellum. The DNA content (per wet tissue) was higher in the hypothalamus and cerebellum. The intracellular concentrations of RNA and tubulin (per DNA) were significantly decreased in the hypothalamus. These values were restored to normal by the administration of L-thyroxine(T4) to rats between 1 and 10 days old. These results demonstrate that fetal nervous tissues are sensitive to changes in thyroid functions. The different manner of response to the hormonal states among the brain areas may reflect that each nervous tissue has a specific critical period for thyroid hormones during development.