Identification of embryonic stem cell–derived midbrain dopaminergic neurons for engraftment

Abstract

Embryonic stem cells (ESCs IntroductionEmbryonic stem cells (ESCs) represent a potentially unlimited source of cells for applications in regenerative medicine. One promising strategy is the derivation of midbrain dopaminergic (DA) neurons for cell replacement therapy in Parkinson disease (PD). Several protocols have been developed to obtain ESC-derived midbrain DA neurons in vitro, though current technologies are far from perfect and cell preparations often contain contaminating non-DA cell populations and potentially tumorigenic cell types (1, 2). Furthermore, despite more than 40 years of research on developing DA cell replacement paradigms, many fundamental questions critical for effective clinical translation have remained unanswered, such as the stage of DA neuron development most appropriate for transplantation; whether other non-dopamine cell types within the graft, such as glia, are necessary for in vivo DA neuron survival and function; and how closely in vitro-generated cells match the properties of authentic midbrain DA neurons generated in vivo. The use of ESC technology has the potential to address all these questions and presents a virtually unlimited source of fully defined DA neurons.One strategy to obtain highly enriched populations of midbrain DA neurons is the purification of ESC-derived progeny prior to transplantation through the use of fluorescence-activated cell

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

“…This finding supports and extends previous work in this area, in which fluorescent transgenes have been used to isolate mDA progenitors that can form functional mDA neurons after transplantation (2,3,5,6). Although the concept of targeting surface antigens is not in itself new, its practical development has been limited by a lack of knowledge of transmembrane proteins with some degree of selectivity for mDA neurons.…”

“…Recent studies have used genetic labeling approaches to demonstrate that such an approach is feasible using fetal ventral mesencephalon (VM) tissue from transgenic mice (2,3) as well as differentiated embryonic stem cell reporter lines (4)(5)(6). Fluorescent protein expression driven by regulatory elements for genes involved in the embryonic development of mDA neurons, including neurogenin2 (Ngn2), Nr4a2 (Nurr1), and Pitx3, allows for isolation of cells within these gene linages through fluorescent activated cell sorting (FACS) and a significant enrichment of mDA neurons in the resulting grafts.…”

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

“…In human embryonic stem cells, it was shown that surface markers that included CD15, CD24, and CD29 could also define specific neural surface biomarker codes for cell sorting using FACS (23). However, more recently it became possible to label and fluorescently detect the expression of marker genes (such as Nurr1 or Pitx3) that indicated specific midbrain dopamine neuron lineages, and this provided a more direct way of sorting and enriching the cells by positive selection (6,24,25). Such efforts led to the first evidence that pure (or nearly pure) dopaminergic neural populations could restore function in parkinsonian animal models (6,24).…”

“…However, more recently it became possible to label and fluorescently detect the expression of marker genes (such as Nurr1 or Pitx3) that indicated specific midbrain dopamine neuron lineages, and this provided a more direct way of sorting and enriching the cells by positive selection (6,24,25). Such efforts led to the first evidence that pure (or nearly pure) dopaminergic neural populations could restore function in parkinsonian animal models (6,24). In particular, Corin expression in the midbrain progenitor cells led to experiments that confirmed that Corin sort selection was an appropriate way to enrich the desirable midbrain dopamine neuron populations (26).…”

“…Over the last decade, attempts to replace missing dopamine neurons have evolved from an innovative and complex fetal dopamine cell transplantation method toward a potential scalable method that depends on stem cell-derived dopamine neurons (2)(3)(4)(5)(6)(7)(8)(9)(10). For functional recovery to occur, the precise midbrain dopamine neuron (denoted A9) lost-which normally connects exactly with the caudate putamen target and inititates movement-needs to be replaced.…”