Project description:Embryonic day (E)12.5 whole murine embryos, E11.5 - E14.5 whole murine embryos, E11.5 - E14.5, post-natal day (P)3 and P35 murine forelimbs, E14.5 brains, and COL1A2-mutant and COL1A2-WT forelimbs were fractionated and specific fractions were analyzed via LC-MS/MS. Aha-enrichment experiments consisted of in vivo protein labeling with azidohomoalanine (Aha) followed by tissue fractionation of the forelimbs and enrichment of labeled ECM proteins from the final IN pellet ('enriched'). 'Unenriched samples', or the background from which newly synthesized proteins were enriched from, were also analyzed via LC-MS/MS.
Project description:Ventral midbrain (VM) dopaminergic progenitor cells derived from human pluripotent stem cells have the potential to replace endogenously lost dopamine neurons and are currently in preclinical and clinical development for treatment of Parkinson’s Disease (PD). However, one main challenge in the quality control of the cells is that rostral and caudal VM progenitors are extremely similar transcriptionally though only the caudal VM cells give rise to dopaminergic neurons with functionality in PD. Therefore, it is critical to develop assays which can rapidly and reliably discriminate rostral from caudal VM cells during clinical manufacturing. Here, we applied shotgun proteomics to search for novel secreted biomarkers specific for caudal VM progenitors compared to rostral VM progenitors and validated key hits by ELISA. From this, we identified novel secreted markers (CPE, LGI1 and PDGFC) significantly enriched in caudal versus rostral VM progenitor cultures, whereas the markers CNTN2 and CORIN were significantly enriched in rostral VM cultures. With this data, we suggest and test in clinical grade samples a panel of coupled ELISA assays that can be applied as a quality control tool for assessing the correct patterning of cells during clinical manufacturing.
Project description:The 5HT system is organized into rostral and caudal populations with discrete anatomical locations and opposite axonal trajectories in the developing hindbrain. 5HT neuron cell bodies in the rostral subdivision migrate to the midbrain and pons and extend ascending projections throughout the forebrain. 5HT cell bodies in the caudal subdivision migrate to the ventral medulla and caudal half of the pons and provide descending projections to the brainstem and spinal cord. Experiment Overall Design: We used microarrays to determine genes expressed by both rostral and caudal 5HT neurons as well as genes that are differentially expressed between rostral and caudal 5HT neurons at E12.5 when axon pathfinding and cell migration are underway. E12.5 neural tubes were isolated from ePet-EYFP embryos and dissected into a rostral domain (mesecephalic flexure to pontine flexure) and a caudal domain (pontine flexure to spinal cord). After cell dissociation (details under growth protocol), cells were subjected to fluorescent activated cell sorting (FACS) to obtain 4 cell populations. R+ = rostral ePetEYFP positive 5HT neurons; R- = YFP negative non-serotonergic cells in the rostral neural tube; C+ = caudal ePetEYFP positive 5HT neurons; C- = YFP negative non-serotonergic cells in the caudal neural tube. 200,000 cells for each of the 4 cell types (R+, R-, C+, C-) were collected for RNA extraction and hybridization to Affymetrix Mouse 430 2.0 arrays.
