Dopaminergic neurons derived from human induced pluripotent stem cells survive and integrate into 6-OHDA-lesioned rats

Abstract

Cell replacement therapy could be an important treatment strategy for Parkinson's disease (PD), which is caused by the degeneration of dopamine neurons in the midbrain (mDA). The success of this approach greatly relies on the discovery of an abundant source of cells capable of mDAergic function in the brain. With the paucity of available human fetal tissue, efforts have increasingly focused on renewable stem cells. Human induced pluripotent stem (hiPS) cells offer great promise in this regard. If hiPS cells can be differentiated into authentic mDA neuron, hiPS could provide a potential autologous source of transplant tissue when generated from PD patients, a clear advantage over human embryonic stem (hES) cells. Here, we report that mDA neurons can be derived from a commercially available hiPS cell line, IMR90 clone 4, using a modified hES differentiation protocol established in our lab. These cells express all the markers (Lmx1a, Aldh1a1, TH, TrkB), follow the same mDA lineage pathway as H9 hES cells, and have similar expression levels of DA and DOPAC. Moreover, when hiPS mDA progenitor cells are transplanted into 6-OHDA-lesioned PD rats, they survive long term and many develop into bona fide mDA neurons. Despite their differentiation and integration into the brain, many Nestin+ tumor-like cells remain at the site of the graft. Our data suggest that as with hES cells, selecting the appropriate population of mDA lineage cells and eliminating actively dividing hiPS cells before transplantation will be critical for the future success of hiPS cell replacement therapy in PD patients.

FIG. 1.

Bright field pictures of (A) undifferentiated human-induced pluripotent stem (hiPS) cells (Stg I), (B) embryonic bodies (Stg II), and (C) neural differentiating hiPS cells (Stg III). The arrow in C indicates rosettes observed in Stg III cells (inset). Scale bar = 100 μm for A and C, 250 μm for B.

FIG. 2.

Expression of neural markers at Stg IV of human-induced pluripotent stem (hiPS) cells. (A) Neural progenitor markers Nestin (red) and Sox2 (green) were detected in many Stg IV hiPS cells, including cells inside rosettes. (B) Dopamine neurons in the midbrain (mDA) lineage markers Aldh1a1 and Lmx1a were expressed in Stg IV hiPS cells. Notice that there were 2 types of Aldh1a1 expressing cells: one was with long processes and inside rosettes or aggregates (arrowhead), the other was flat and outside aggregates (arrow). (C) Similar to human embryonic stem (hES) cells, mDA-specified hiPS cells labeled by Lmx1a also expressed TrkB on their surface. Dapi was used to counterstain all the nuclei in the field (A–C). Scale bar = 50 μm for A–C. (D–F) Higher magnification confocal images of A–C, respectively.

FIG. 3.

RT-PCR and immunocytochemical analyses of DA-related markers in late stages of human-induced pluripotent stem (hiPS) cells differentiation in culture. Many Aldh1a1+/TH+ dopamine neurons in the midbrain (mDA) neurons [(A) fluorescent microscope image, (B) confocal microscope image] and some Lmx1a+/Aldh1a1+ earlier step cells in the mDA differentiation (C) were observed at Stg V. Dapi was used to counterstain all the nuclei in the field. The yield of TH+ expressing cells in Stg V-differentiated hiPS cells is similar as hES cells. Scale bar = 50 μm for A and C. (D) DA-related genes are differentially expressed at stages IV–V of hiPS cell differentiation detecting by RT-PCR. (E) StgV of both H9 and hiPS cells express GLAST, but not other neurotransmitter system genes. (F) Significantly higher levels of DA and DOPAC were detected in Stg V hiPS cells and H9 cells as well.

FIG. 4.

Immunofluorescence analysis of DA-related markers in stage IV human-induced pluripotent stem (hiPS) cells 6 weeks after transplantation. Most survived transplanted cells in the graft as detected by human nuclear antigen (HNA) are Nestin+ neural progenitor cells (A). Some are still Sox2+ neural progenitor cells (B). Occasionally proliferating Ki67 cells were found in the HNA+ grafted cells (C). Arrowhead pointed area is shown at a higher magnification in the inset. Clusters of Lmx1a+/Aldh1a1+, Aldh1a1+ precursor cells (D) were found in the grafts. Insets of D show the majority of these DA precursor cells were HNA+. Mature TH+/Aldh1a1+ neurons were also detected (E and F). Inset of E shows a grafted TH+/HNA+ neuron was surrounded by host cells (HNA–). Scale bar = 50 μm.