Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

Abstract

An important challenge for the continued development of cell therapy for Parkinson's disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a "FACS-array" approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.Parkinson's disease | cell sorting | Alcam | microarray | transplantation

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

“…As a control, we implanted 250,000 mDA neurons that were generated on 2D Matrigel-coated surfaces as previously reported1. Six weeks post-implantation, a time point at which previous mDA neuron transplantation studies begin to report functional improvements in PD rat models36, we sacrificed the animals and investigated graft survival (Figs 4 and S8). TH and FOXA2 expression was observed in the HNA+ surviving cells among both the 3D (4a–j) and 2D (4k–n) generated mDA neuron groups.…”

Sorting the wheat from the chaff in dopamine neuron-based cell therapies

“…These authors also identified the selectable surface marker Alcam (26). In a report published in PNAS, Bye et al (27) demonstrate another strategy that also obtained evidence of at least one of these markers (Alcam). Bye et al base their analysis on mRNA expression from sorted neurogenin2-GFP progenitor cells (28) and then comparatively by using the GFP − fraction (nondopaminergic) cell populations expressing Lmx A1 GFP (11), and by post hoc analysis subtracted less-relevant mRNA expression.…”

“…This arithmetic provided support for a separate analysis and selection of transmembrane proteins expressed only on developing midbrain dopamine neurons. Bye et al (27) prioritized four surface transmembrane receptor marker candidates: Alcam, Chl1, Gfra1, and Igsf8. The authors confirmed and replicated the findings of Doi et al (26), that Alcam was expressed widely throughout the midbrain dopaminergic region.…”

“…The authors confirmed and replicated the findings of Doi et al (26), that Alcam was expressed widely throughout the midbrain dopaminergic region. Using FACS, Bye et al (27) were able to sort by the Alcam marker and obtain an almost 30-fold enrichment in midbrain dopamine neurons; however, they lost a large fraction of these cells because of the cell-sorting procedure. When transplanting such enriched cell populations in a rodent model of Parkinson's disease, Bye et al found that the surviving dopamine neurons had the capacity to reinnervate the host striatal target brain territory and restore function (27).…”

Mass Spectrometry-Based Identification of Extracellular Domains of Cell Surface N-Glycoproteins: Defining the Accessible Surfaceome for Immunophenotyping Stem Cells and Their Derivatives

“…While approximately 370 antigens are targeted by monoclonal antibodies to cluster of differentiation (CD) molecules [8], this represents only a fraction of all molecules present on the cell surface (i.e., the surfaceome) and CD molecule screening has had limited success for nonhematopoietic stem cells [9–11]. Alternative approaches have been employed to evaluate the cell surface protein landscape based on transcriptomics [12], and physical and affinity enrichment strategies coupled with proteomics [13, 14], although each has limitations. Transcript abundance does not always correlate with protein abundance on the cell surface, transcriptomic data fail to inform subcellular localization, and the precise location of many membrane proteins are yet unannotated.…”