Interspecies chimerism with human embryonic stem cells generates functional human dopamine neurons at low efficiency

Interspecies chimeras offer great potential for regenerative medicine and creation of human disease models. Whether human pluripotent stem cell (hPSC) derived neurons in an interspecies chimera can differentiate into functional neurons and integrate into host neural circuity is not known. Here we show, using Engrailed 1 (En1) as a development niche that human naïve-like ES cells can incorporate into embryonic and adult mouse brains. Human-derived neurons including tyrosine hydroxylase (TH) positive neurons integrate into the mouse brain at low efficiency.  These TH-positive neurons have electrophysiologic properties consistent with their human origin. Additionally, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils.  Optimization of human/mouse chimeras could be utilized to study human neuronal differentiation and human brain disorders., Culture of Human ES Cells
Human ES cell line RUES1 and RUES2 were obtained from the WiCell Research Institute. The formulation of hESC medium is: DMEM/F12 supplemented with 20% knockout serum replacement (KSR), 1% GlutaMAX, 1% nonessential amino acids, 0.1 mM β-Mercaptoethanol, and 10 ng/ml bFGF. The primed hES cells were cultured on a feeder layer of irradiated mouse embryonic fibroblast (MEF) cells in hESC medium under 5% CO2 at 37 °C. To maintain primed hESCs in an undifferentiated state, the irradiated feeder cells were freshly prepared one day before the passage. Primed hESCs were routinely passaged every 5-7 days at a split ratio of 1:3 by a PBS wash followed by Collagenase IV. 
hES cells labelling
For chimeras, RUES1 or RUES2 were made to express a CAG-GFP or CAG-dsRed expression cassette by piggyBac transposon transposition. The CAG-GFP or CAG-dsRed plasmid (2.5 µg) and Transposase (2.5 µg) were transfected into ES cells using a nucleofector device (Lonza). Four days after tra..., , # Interspecies chimerism with human embryonic stems cells generates functional human dopamine neurons at low efficiency

**In this study, we used Engrailed 1 (En1) as a development niche that human naïve-like ES cells can incorporate into the embryonic and adult mouse brain. Human-derived neurons including tyrosine hydroxylase (TH) positive neurons integrate into the mouse brain at low efficiency.  These TH positive neurons have electrophysiologic properties consistent with their human origin. Additionally, these human-derived neurons in the mouse brain accumulate pathologic phosphorylated α-synuclein in response to α-synuclein preformed fibrils.** 
 
**Description of the data and file structure**

**Figure Files**
1.      Figure 1: Chimeric contribution of human ES RUES1-derived cells in En1 knockout mouse brain  

a.      Figure 1a: Summary of blastocyst injections collected chimeric embryos and offspring

b.      Figure 1b: Representative bright field image of mouse embryo at E10.5...