- Generation of human induced Pluripotent Stem cells (hiPSCs), neuronal differentiation of hiPSCs.
- Xenograft transplantation of human neuronal stem cells into specific brain areas of NOD-SCID mice.
- Generation and network analysis of large-scale mRNA and microRNA expression data, RNAseq, Exome.
- Neuronal differentiation of hiPSCs, particularly into Radial Glial cells
- Onset of brain tumor formation
- In-vivo brain tumor modeling
- Nanotechnology – diagnostic mapping of Radial Glial cells in the human brain by MRI
Induced pluripotent stem cells
Study of embryonic stem cell (ESC) differentiation is of enormous interest given the therapeutic potential in cell replacement therapy. However, there are well known ethical barriers related to the use of human embryos as the source of ESCs as well as “technical” ones related to generation of patient- specific ESCs. Induced pluripotent stem cell (iPSC) technology, which is based on reprogramming of differentiated somatic cells into a pluripotent state, could evade those barriers as iPSC can be derived with a specific desired genetic background, including patient-specific iPSC for disease models and for transplantation therapies, without the problems associated with immune rejection.
Induction of pluripotency in adult human fibroblasts was originally achieved by Dr. Yamanaka and colleagues by enforced expression of four transcription factors: KLF4, c-MYC, OCT4, and SOX2, using retroviral vectors. Since we are generating the iPSC lines for the disease modeling, instead of the retroviral vectors, which integrate into genome during the reprogramming and potentially could cause undesirable gene alterations, we are using non-integrative Sendai viral vectors.
iPSC colony (30 days after the reprogramming)
Radial Glia cells
The development and function of the nervous system is very complex and little understood. One of the biggest limitations in research of nervous system disorders is a lack of laboratory systems to study the course of disease and to identify potential drug targets. Human neuronal or brain cells typically cannot be examined experimentally until surgical or postmortem biopsy samples are obtained. These limitations become even more severe in the case of pediatric disorders. Derivation of neuronal stem cells (NSC) can provide an efficient route to valuable disease models. Radial Glia (RG) cells are thought to be the progenitor cells for adult NSC, neurons, basal progenitors, astrocytes and oligodendrocytes, in addition to being responsible for the majority of neurogenesis in the developing brain. RG processes provide architectural support for the migration of newly generated neurons by forming scaffolds that span the central nervous system mantle region. In addition to being responsible for the majority of neurogenesis in the developing brain RG cells are not only of relevance to neurodegenerative diseases, and spinal injuries, but to brain tumors as well.
Recently we reported a new approach that allowed us to derive large amounts of RG cells from hESC, and from hiPSC lines. We demonstrated that RG cells orthotopically transplanted to the motor cortex of 8-week old immunocompromised NOD-SCID mice can differentiate into functionally active, mature-appearing pyramidal and serotonergic neurons. The ability to generate these cells in amounts is very significant because it enables a wide range of experimentation that otherwise could not be done.
Sergey Malchenko, Jianping Xie, Maria de Fatima Bonaldo, Elio F. Vanin, Bula Bhattacharyya,Vasily Galat, William Goossens, Richard E.B. Seftor, John Crispino, Richard Miller, Martha C. Bohn, Mary J.C. Hendrix and Marcelo B. Soares.2014. Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies. GENE 534 400-407.
The differentiation of GFP positive RG cells into mature pyramidal and serotonergic neurons after being injected into motor cortex of NOD-SCID mice (3 weeks post-injection): phase contrast anti-GFP DAB staining (63×magnification) (A), Co-staining of DAPI and GFP, white arrows indicate the presence of dendritic spines (63 x magnifications) (B). Electrophysiological recording from GFP positive RG cells engrafted into mice hippocampal slice cultures. The voltage clamps recordings from these cells demonstrated TTX-sensitive voltage-dependent Na currents (C) blocked by TTX (500 nM) (D).
In-vivo brain tumor modeling
We also showed that orthotopic transplantations of the RG cells to the subventricular zone of the 3rd ventricle – but not to other transplantation sites – of the brain in immunocompromised NOD-SCID mice, gave rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. The tumors are highly invasive, and they effectively recruit mouse endothelial cells for angiogenesis.
In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal RG cells to a malignant phenotype.
Sergey Malchenko, Simone Treiger Sredni, Atsushi Kasai, Kazuki Nagayasu, Kaoru Seiriki, Jianping Xie, Naira Margaryan, Hitoshi Hashimoto, Rishi Lulla, Lauren Pachman, Herbert Y. Meltzer, Mary J.C. Hendrix and Marcelo B. Soares. 2015. A Mouse Model of Radial Glial Cell-Derived Primitive Neuroectodermal Tumors. PLOS ONE Mar 31;10(3):e0121707.
3D imaging of the mice brain using a confocal microscope at one month A- or two months B- post—injection. Top left panels reconstitute images of the brains placed horizontally, top right panels reconstitute coronal sections at the indicated lines 1–3. Bottom left panels reconstitute 3D image of the brains oriented as shown by the red plot lines. The section images at the white dotted lines shown at the bottom right panels. A—anterior; P—posterior; R-right; L—left.
Nanotechnology – diagnostic mapping of Radial Glial cells in the human brain by MRI
The dynamic distribution of RG in the human brain is still largely unknown despite its importance in the development and homeostasis of Central Nervous System and likely relevance in the onset of brain tumors, dementias, acquired and congenital neurodegenerative diseases. Therefore, the ability to safely monitor the dynamic distribution of RG in the brain is highly desirable and likely to prove of significant diagnostic value. Thus, there is an obvious need to develop a non-invasive in-vivo diagnostic imaging approach – such as magnetic resonance imaging (MRI) – that might enable dynamic mapping of RG, i.e. spatially and temporally, in the human brain. Adult stem cells such as mesenchymal stem cells and NSCs have received considerable attention for use as magnetic nano-structure (MNS) carriers. Both cell types can be loaded with MNSs without affecting their normal cellular function, and can be tracked using MRI after implantation into rats. However, the technical challenges involved in the isolation of NSCs in sufficient amounts for experimentation remain a major obstacle to the advancement of nanotechnology in the field of NSC biology.
Our approach to derive large amounts of RG cells created an opportunity for the establishment of new collaborations that could not have been sought before. One of such collaborations is with Dr. Vinayak Dravid from the Northwestern International Institute for Nanotechnology. We are taking advantage of our ability to derive unrestricted amounts of RG cells to experiment with nanoparticles of different compositions to attain conditions under which RG cell specificity may be attained. This would enable diagnostic mapping of RG cells in the human brain by MRI, in normal brain and in the brain of individuals affected with neurodegenerative diseases. Next, we seek to identify differences between normal and malignant RG cells with the goal of using nanoparticles to specifically target and kill malignant RG cells that would otherwise function as brain tumor stem cells.
Current repository of iPSCs
- 4 iPSC lines developed from peripheral blood of patients with schizophrenia.
- 6 iPSC lines developed from peripheral blood of patients with astrocytoma (2), germinoma, medulloblastoma (2), and low-grade glioma.
- 6 iPSC lines developed from peripheral blood of patients with JDM and age/gender matched “control” group.
Current repository of Radial Glia stem cells
- 1 RG line developed from iPSC of a healthy child
- 2 RG lines developed from iPSC of patients with schizophrenia
- 5 RG lines developed from iPSC of patients with astrocytoma, germinoma, medulloblastoma, and low-grade glioma.