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Recent Findings

Pregnant female mice were exposed to heroin, organophosphate or other substances. Their offspring showed, upon maturity, an abolishment of cholinergic receptor-induced activation/translocation of PKCγ in the septohippocampal cholinergic innervation, paralleled with defects in the hippocampus-related Morris and eight-arm maze behaviors.
Understanding the synaptic mechanism of the behavioral defects enabled reversal of the neurobehavioral teratogenicity using the various therapies described above:
Further studies suggest that one major mechanism by which the transplanted cells exerted their therapeutic action is by the induction of neurogenesis and synaptogenesis in the teratogen-impaired brain. Further studies demonstrated impairment of neurogenesis (doublecortin and neurogenesis-related genes) after prenatal exposure to teratogen (chlorpyrifos). Transplantation of MSC restored normal neurogenesis. 
In the chick model, inc​ubated eggs were injected with various teratogens. The hatched chicks showed marked defects in their imprinting behavior, which was correlated with abolishment of cholinergic-induced activation/translocation of PKCγ in their IMHV (IMM) nucleus. Additionally, neurogenesis was impaired as attested to by reduced doublecortin labeling and neurogenesis-related genes. Stem cells transplanted to the chick embryo via the blood vessels attached to the chorio-allantoic membrane reach the embryonic brain and restored normal neurogenesis. 
Currently, we are testing the hypothesis, in both models, that the changes described may be regulated by the teratogen-induced epigenetic alterations. 
The study of the reversal of heroin and chlorpyrifos (an organophosphate)-induced neurobehavioral teratogenicity serves as examples: NSC derived from normal developing brains (Fig. 1) were grafted into the hippocampus of mice offspring who were exposed prenatally to the teratogens and showed hippocampus related defects in behavior and PKC activation/translocation.
Fig. 1 Differentiation of ES-NSC dissociated precursor cells into neural lineage cells.
A. ES-NSC grown in the presence of the growth factors EGF and bFGF are positive for neural marker- Nestin. B-D. Growth factor removal promoted the differentiation of ES-NSC into neurons, oligodendrocyte and astrocytes; B. NF160 positive differentiated neurons, B’. High proportion of NF positive cells exhibit cholinergic marker ChAT. C. O4 positive oligodendrocytes. D. GFAP positive astrocytes. E. PCR Expression of Neural genes with and without growth factor. Each band of the neural lineage genes tested was normalized in relation to the house keeping gene GAPDH. Size bars: A-D: 100 μm. F. Quantitative representation of the gene expression findings with and without (w/o) growth factors (GF).
The transplanted cells survived and differentiated in the host hippocampus as shown in Fig. 2. Offspring exposed prenatally to heroin had at maturity deficits in Morris maze behavior (A) and in the mechanistically-related absolute abolishment of cholinergic receptor-induced activation/translocation of hippocampal PKCγ (B). Transplantation of NSC into the impaired hippocampus reversed the neurobehavioral defects (Fig. 3). 
Fig. 2
A. Grafted BrdU-labeled NSC identified in the brain of the teratogen-impaired brain by immunofluorescent staining (green). Some of the transplanted cells acquire markers of specific neural lineages. In the present case double labeling BrdU+, GFAP+ astrocytes (red, confocal microscopy).
B. CM-DiI-Labeled adult (subventricular zone-derived) NSC post transplantation were shown to differentiate to cholinergic neurons expressing ChAT (arrows, DiI - red, DAPI – blue).
Fig 3.
A. Transplantation of NSC reversed the prenatal heroin-induced deficits in Morris maze behavior. Numbers represent the time spent to reach the platform (mean±SEM).
Control (pooled) = Prenatal control – postnatal medium or NSC.
H-C = Prenatal heroin - postnatal medium.
H-NP = Prenatal heroin - postnatal NSC.
B. Transplantation of NSC reversed the heroin-induced desensitization of hippocampal PKCg.
C-C = Prenatal control – postnatal medium
C-NP = Prenatal control - postnatal NSC
H-C = Prenatal heroin - postnatal medium.
H-NP = Prenatal heroin - postnatal NSC
We hypothesize that one major mechanism by which the stem cells exert their therapeutic action is the induction of neurogenesis in the host brain. Indeed, transplantation of NSC induced increased production of endogenous cells in the brain (Fig. 4.)



Neurogenesis in the dentate gyrus in transplanted versus control heroin-exposed mice. Few BrdU-labeled cells were observed (light stain, Alexa 488) in the granular cell layer of mice exposed prenatally to heroin and sham operated (A), but significantly more in mice transplanted with NSC (B).
Chick model:
Chicks were exposed to various teratogens prehatch (in the incubating egg). After hatching, they showed alterations in PKCγ in the IMHV nucleus (Fig. 5A) and concomitant deficits in the IMHV-related imprinting behavior (Fig. 5B).
Fig. 5
A. Preference ratio in control chicks and chicks with prehatch exposure to nicotine, chlorpyrifos or heroin (means ±SEM). ***P<0.001, * P<0.05) versus respective controls (ANOVA). P<0.001 for the difference from no preference (score = 0.5).
B. Effect of prehatch exposure to chlorpyrifos basal membrane levels of PKC isoforms. Data represent means and standard errors of the differences from control levels (means ±SEM). * P<0.05, **P<0.01 versus zero change.
Subsequently we have established a model for transplantation of stem cells into the brain of the chick embryo (Fig. 6). Prehatch- exposure to chlorpyrifos impaired neurogenesis as demonstrated with doublecortin labeling. Transplantation of MSC to the chick embryo restored normal neurogenesis (Fig. 7). 
Fig 6. IV transplanted stem cells in the chick embryo migrated to its brain and survived. CM-Dil labeled cells were IV transplanted into E13 chick embryos and the cells were tracked in posthatch 1 chick embryos' brains. Transplanted cells have migrated along the blood vessels and reached the third ventricle of the chick embryos' brains.
Fig. 7. Reversal of neuroteratogenicity in a chick-chlorpyrifos (CPF) model: Transplantation of MSC reversed prehatch chlorpyrifos-induced alterations in neurogenesis (doublecortin labeling)
The X axis (0.0) represents the transplanted (media) group (N=7). Where the bars represent proportional differences from Control Sham C TR, control transplanted (with MSC); CPF SH, CPF sham; CPF TR, CPF transplanted. N = number of brains.

Recent studies identified neurogenesis-related genes that were down-regulated by the prehatch exposure to chlorpyrifos as is shown in Fig. 8. Transplantation of MSC to the chick embryo restored normal expression of these genes. 

Fig. 8


Fig. 8. The effects of MSC transplantation on neurogenesis-related gene expression in the lateral striatum area following prenatal exposure to CPF.
Left / Right – sides of brain; numbers at the bottom of each column = N. TR - transplanted
* P<0.05; ** P<0.01; *** P<0.001.