Engraftment of Mouse Embryonic Stem Cells Differentiated by Default Leads to Neuroprotection, Behaviour Revival and Astrogliosis in Parkinsonian Rats

Abstract

We report here protection against rotenone-induced behavioural dysfunction, striatal dopamine depletion and nigral neuronal loss, following intra-striatal transplantation of neurons differentiated from murine embryonic stem cells (mES). mES maintained in serum free medium exhibited increase in neuronal, and decrease in stem cell markers by 7th and 10th days as revealed by RT-PCR and immunoblot analyses. Tyrosine hydroxylase, NURR1, PITX3, LMX1b and c-RET mRNA showed a significant higher expression in differentiated cells than in mES. Dopamine level was increased by 3-fold on 10th day as compared to 7 days differentiated cells. Severity of rotenone-induced striatal dopamine loss was attenuated, and amphetamine-induced unilateral rotations were significantly reduced in animals transplanted with 7 days differentiated cells, but not in animals that received undifferentiated ES transplant. However, the ratio of contralateral to ipsilateral swings in elevated body swing test was significantly reduced in both the transplanted groups, as compared to control. Striatal grafts exhibited the presence of tyrosine hydroxylase positive cells, and the percentage of dopaminergic neurons in the substantia nigra was also found to be higher in the ipsilateral side of 7 days and mES grafted animals. Increased expression of CD11b and IBA-1, suggested a significant contribution of these microglia-derived factors in controlling the limited survival of the grafted cells. Astrocytosis in the grafted striatum, and significant increase in the levels of glial cell line derived neurotrophic factor may have contributed to the recovery observed in the hemiparkinsonian rats following transplantation.

Embryonic Stem Cells Derived Neuron Transplantation Recovery in Models of Parkinsonism in Relation to Severity of the Disorder in Rats

“…32,37 When equal numbers (2 · 10 5 cells) of these cells differentiated for 7 days were transplanted in the striatum of animals with two different doses (16 and 32 nmol) of 6-OHDA-or MPP + -infused animals, we observed the extent of recovery in terms of amphetamine-and apomorphine-induced rotations. Striatal DA levels were better in animals that received a lower dose of the toxin, irrespective of the type of toxin used.…”

“…Rotations were counted by persons who were trained in the behavioral assessments, but were blinded to the treatments administered. [31][32][33] For characterization of the lesion severity (i.e., dose-dependency of the neurotoxins used), amphetamine and apomorphine rotations were done respectively on the 46 th and 48 th days.…”

“…Mouse embryonic stem cells (mESCs) were differentiated in serum-free medium for 7 days, as detailed by Tripathy et al 32 In short, D3 mESCs (American Type Culture Collection [ATCC] no. CRL11632; Manassas, VA) maintained in Dulbecco's modified Eagle Medium (DMEM; GibcoInvitrogen) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM glutamine (Gibco-Invitrogen), and 1000 Units of non-essential amino acids, 100 units of penicillin and 100 mg streptomycin (Gibco-Invitrogen), 0.7% b-mercaptoethanol (MP Biomedicals, Solon, OH), and 1000 units of leukemia inhibitory factor (LIF; Millipore, Billerica, MA).…”

“…32,34 Animals were anesthetized on the 50 th day after toxin infusion, perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4) initially, followed by 4% paraformaldehyde perfusion. The brain was dissected out, kept in the fixative overnight, and transferred to 30% sucrose solution.…”

“…The sections were mounted in DPX Mountant, following dehydration with graded alcohol and clearing in xylene. 32,34 The anti-rabbit IgG antibody-Alexa Flour 564-tagged sections were mounted on slides with Antifade (UltraCruz TM , sc-359850, Santa Cruz/ProLong Gold antifade, Invitrogen) mounting medium and viewed in an epifluorescence inverted microscope (Karl Ziess, Germany).…”