Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells

A. Sánchez-Danés, A. Consiglio, Y. Richaud, I. Rodríguez-Pizà, B. Dehay, M. Edel, J. Bové, M. Memo, M. Vila, A. Raya, J.C. Izpisua Belmonte
Human Gene Therapy. 2012-01-01; 23(1): 56-69
DOI: 10.1089/hum.2011.054

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Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) offer
great hope for in vitro modeling of Parkinson’s disease (PD), as well as for
designing cell-replacement therapies. To realize these opportunities, there is an
urgent need to develop efficient protocols for the directed differentiation of
hESC/iPSC into dopamine (DA) neurons with the specific characteristics of the
cell population lost to PD, i.e., A9-subtype ventral midbrain DA neurons. Here we
use lentiviral vectors to drive the expression of LMX1A, which encodes a
transcription factor critical for ventral midbrain identity, specifically in
neural progenitor cells. We show that clonal lines of hESC engineered to contain
one or two copies of this lentiviral vector retain long-term self-renewing
ability and pluripotent differentiation capacity. Greater than 60% of all neurons
generated from LMX1A-engineered hESC were ventral midbrain DA neurons of the A9
subtype, compared with ∼10% in green fluorescent protein-engineered controls, as
judged by specific marker expression and functional analyses. Moreover, DA neuron
precursors differentiated from LMX1A-engineered hESC were able to survive and
differentiate when grafted into the brain of adult mice. Finally, we provide
evidence that LMX1A overexpression similarly increases the yield of DA neuron
differentiation from human iPSC. Taken together, our data show that stable
genetic engineering of hESC/iPSC with lentiviral vectors driving controlled
expression of LMX1A is an efficient way to generate enriched populations of human
A9-subtype ventral midbrain DA neurons, which should prove useful for modeling PD
and may be helpful for designing future cell-replacement strategies.

DOI: 10.1089/hum.2011.054
PMCID: PMC3472681
PMID: 21877920 [Indexed for MEDLINE]

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