Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells

Abstract

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%-95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.

Fig. 1.

Lmx1a promotes generation of mesDA^mES neurons. (A) Lmx1a is expressed in DA progenitors and postmitotic TH⁺ neurons at embryonic d 10.5 and 12.5 in mouse ventral MB. (B) NesE-Lmx1a and NesE-eGFP mESCs were differentiated and analyzed. At 4 DDC, >85% Nestin⁺ cells derived from NesE-Lmx1a and NesE-eGFP cells expressed Lmx1a or eGFP, respectively. At 12 DDC, few TuJ1⁺ cells derived from NesE-eGFP cells expressed TH whereas >75% of TuJ1⁺ cells derived from NesE-Lmx1a expressed TH and other mesDA markers. Generation of 5-HT neurons was repressed in NesE-Lmx1a cultures. (C) Quantification at 12 DDC. (D) Quantification of NesE-Lmx1a and NesE-eGFP ESCs differentiated according to the 5-stage protocol. More than 60% of Lmx1a-induced TH⁺ neurons co-expressed Lmx1a, Nurr1, and Pitx3 at stage 5 d 10 and DAT at d 20. (See Fig. S1 for expression patterns.) (C–D) Error bars indicate SD, n = 5.

Fig. 2.

Lmx1a-induced mesDA^mES neurons show the same physiological properties as mesDA neurons in vivo. (A) Electrophysiology of randomly chosen neurons showed that 60% of Lmx1a-induced neurons, but only 10% of control neurons, showed properties of mesDA neurons (See SI Text for additional information). (B) HPLC analysis showed a 4-fold increase in DA and DOPAC content in NesE-Lmx1a-induced neurons compared with NesE-eGFP-derived neurons.

Fig. 3.

Lmx1a-induced progenitors give rise to grafts containing many mesDA^mES neurons following transplantation into rat striatum. (A) Immunohistochemistry for TH illustrates the typical dense staining of DA fibers in the intact striatum; (B) almost complete loss of TH fibers is seen in 6-OHDA lesioned striatum; (C) there is substantial graft-derived re-innervation of the lesioned striatum 4 weeks after transplantation (arrowheads, graft core). (D) The boxed area in C is shown at larger magnification. The innervation of the host striatum is most dense in close proximity to the graft and extends over large distances. (E) Typical appearance of part of an intra-striatal graft and associated fiber outgrowth revealed by TH staining. The graft core (Left) contains many intensely stained TH⁺ neurons, which innervate the adjacent host striatum and form a dense terminal network (Right). (F) Most TH⁺ neurons have a large size and elongated shape typical for mesDA neurons. (G–I) Virtually all grafted mesDA^mES neurons co-expressed TH and Pitx3. The vast majority also expressed Nurr1 (J) and En1/2 (K) (boxed areas in J and K depicted as single colors illustrating cytoplasmic and nuclear localization of TH or transcription factors, respectively). (L) Many of the TH⁺ neurons co-expressed VMAT2 (boxed area depicted as single colors to show the overlap and cytoplasmic distribution). (M) Despite large numbers of GABA⁺ neurons, there were only very rare examples of overlap between TH and GABA. The grafts contained different mesDA neuronal subtypes as indicated by co-expression of Girk2 (N and O) or Calbindin (P and Q) in TH⁺ cells (O and Q). Boxed areas shown at greater magnification illustrating overlap between TH and Girk2/Calbindin. (Scale bars, 500 μm in A–C; 200 μm in E and M–Q; 100 μm in F; 50 μm in G–I and J–L.)

Fig. 4.

Grafting of PSA-NCAM-sorted NesE-Lmx1a-derived neurons eliminates overgrowth. (A) Eleven DDC NesE-Lmx1a cells were sorted and transplanted into the striatum of 6-OHDA lesioned neonatal rats. (B) Most of the TH⁺ neurons had a large size and elongated shape typical of mesDA neurons. (C) M2/M6 staining showing the size of the graft. (D) The majority of the transplanted neurons (HuC/D⁺ cells) expressed TH. (E–H) Most of the grafted neurons co-expressed TH and other mesDA markers: Pitx3 (E), Nurr1 (F), En1/2 (G), and VMAT2 (H). There were few 5-HT (I) and GABA (J) neurons in the graft.

Fig. 5.

Lmx1a promotes mesDA^hES neurons in differentiating hESCs. hESC-derived neuroepithelial progenitors were infected with lentiviral (L) vectors carrying Lmx1a or eGFP and analyzed at day 30 to 40 pt. (A) In L-Lmx1a-infected cultures, >50% of TuJ1⁺ neurons co-expressed TH at d 30 pt, compared with 25% in L-eGFP-infected cultures. Most TH⁺ neurons co-expressed mesDA markers, e.g., Lmx1b, Pitx3, Nurr1, and DAT, whereas markers for 5-HT neurons were suppressed. Few TH⁺ neurons derived from L-eGFP-infected cells co-expressed mesDA markers. (B) Differentiation scheme. (C) Quantification of marker expression. Error bars indicate SD, n = 4.