Selective Generation of Dopaminergic Precursors from Mouse Fibroblasts by Direct Lineage Conversion.
ABSTRACT: Degeneration of midbrain dopaminergic (DA) neurons is a key pathological event of Parkinson's disease (PD). Limited adult dopaminergic neurogenesis has led to novel therapeutic strategies such as transplantation of dopaminergic precursors (DPs). However, this strategy is currently restrained by a lack of cell source, the tendency for the DPs to become a glial-restricted state, and the tumor formation after transplantation. Here, we demonstrate the direct conversion of mouse fibroblasts into induced DPs (iDPs) by ectopic expression of Brn2, Sox2 and Foxa2. Besides expression with neural progenitor markers and midbrain genes including Corin, Otx2 and Lmx1a, the iDPs were restricted to dopaminergic neuronal lineage upon differentiation. After transplantation into MPTP-lesioned mice, iDPs differentiated into DA neurons, functionally alleviated the motor deficits, and reduced the loss of striatal DA neuronal axonal termini. Importantly, no iDPs-derived astrocytes and neoplasia were detected in mouse brains after transplantation. We propose that the iDPs from direct reprogramming provides a safe and efficient cell source for PD treatment.
Project description:Transplantation of dopaminergic precursors (DPs) is a promising therapeutic strategy of Parkinson's disease (PD). However, limited cell source for dopaminergic precursors has become a major obstacle for transplantation therapy. Our group demonstrated previously that mouse fibroblasts can be reprogrammed into induced dopaminergic precursors (iDPs) with high differentiation efficiency. In the current study, we hypothesized that a similar strategy can be applied to generate human iDPs for future cell therapy of PD. We overexpressed transcription factors Brn2, Sox2, and Foxa2 in human fibroblasts and observed formation of neurospheres. Subsequent characterization of the precursor colonies confirmed the generation of human induced dopaminergic precursors (hiDPs). These hiDPs were capable of self-renewal, proliferation, and differentiation. The hiDPs demonstrated high immunoreactivity for neural progenitor markers and high levels of gene expression for ventral mesencephalon-related neural progenitor markers such as Lmx1a, NIKX6.1, Corin, Otx2 and Mash1. Furthermore, the hiDPs could be differentiated into dopaminergic neurons with ˜80% efficiency, which significantly increased major functionally relevant proteins such as TH, DAT, AADC, Lmx1B, and VMAT2 compared to hiDPs. Additionally, hiDPs are more dopaminergic progenitor-restricted compare to those hiDP-like cells reprogrammed only by Brn2 and Sox2. Together, these results suggest that hiDPs with high differentiation efficiency can be generated by direct lineage reprogramming of fibroblasts with transcription factors Brn2, Sox2, and Foxa2. These hiDPs may serve as a safe and effective cell source for transplantation treatment of PD.
Project description:Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson's disease. However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. Here, we show that human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, CORIN. We induced DA neurons using scalable culture conditions on human laminin fragment, and the sorted CORIN(+) cells expressed the midbrain DA progenitor markers, FOXA2 and LMX1A. When transplanted into 6-OHDA-lesioned rats, the CORIN(+) cells survived and differentiated into midbrain DA neurons in vivo, resulting in significant improvement of the motor behavior, without tumor formation. In particular, the CORIN(+) cells in a NURR1(+) cell-dominant stage exhibited the best survival and function as DA neurons. Our method is a favorable strategy in terms of scalability, safety, and efficiency and may be advantageous for clinical application.
Project description:By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3? inhibitor) to induce the midbrain floor plate (FP) progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for the generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells and induced pluripotent stem cells (PSCs). We found that CHIR99021 specifies diencephalon to hind brain fates in a concentration-dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain FP progenitors, expressing Corin, En1, FoxA2, and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of tyrosine hydroxylase, Lmx1a/b, FoxA2, FoxP1, Nurr1, and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy.
Project description:Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (mDA) neurons for cell replacement therapy for Parkinson's disease (PD). However, iPSC-derived donor cells inevitably contain tumorigenic or inappropriate cells. To eliminate these unwanted cells, cell sorting using antibodies for specific markers such as CORIN or ALCAM have been developed, but neither marker is specific for ventral midbrain. Here, we employed a double-selection strategy for cells expressing both CORIN and LMX1A::GFP and report a novel cell surface marker to enrich mDA progenitors, LRTM1. When transplanted into 6-OHDA-lesioned rats, human iPSC-derived LRTM1+ cells survived and differentiated into mDA neurons in vivo, resulting in significant improvement in motor behavior without tumor formation. In addition, LRTM1+ cells exhibited efficient survival of mDA neurons in the brain of an MPTP-treated monkey. Thus, LRTM1 can provide a powerful tool for efficient and safe cell therapy for PD patients. Overall design: Differentiated mouse ESC-derived neural progenitors just after sorting (day9 CORIN+LMX1A::GFP+, day9 CORIN-LMX1A::GFP+) and mouse fetal ventral mesencephalon on E11.5 (CORIN+, CORIN-) were subjected to RNA extraction and hybridization on Affymetrix microarrays.
Project description:Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells may inevitably contain tumorigenic or inappropriate cells. Purification of neural progenitor cells or DA neurons as suitable donor cells has been attempted, but the isolation of DA progenitor cells derived from human pluripotent stem cells has so far been unsuccessful. Here we show human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, Corin. we were able to develop a method for 1) scalable DA neuron induction on human laminin fragment and 2) sorting DA progenitor cells using an anti-Corin antibody. Furthermore, we determined the optimal timing for the cell sorting and transplantation. The grafted cells survived well and functioned as midbrain DA neurons in the 6-OHDA-lesioned rats, and showed minimal risk of tumor formation. The sorting of Corin-positive cells is favorable in terms of both safety and efficiency, and our protocol will contribute to the clinical application of human iPSCs for Parkinson’s disease. Differentiated human iPSC-derived neural progenitors just after sorting (day12 unsorted, day12 Corin+) and dopaminergic progenitors after an aggregation culture (day28 and day42, unsorted and day12-sorted, respectively), and human fetal ventral mesencephalon and dorsal mesencephalon (gestational age of 7.5 weeks) were subjected to RNA extraction and hybrdization on Affymetrix microarrays. Each sample except for human mesencephalon, undifferentiated iPSC, and day12-unsorted, day42-sample has 3 or 4 repeats.
Project description:During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2(+)Corin(+) cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2(+)Corin(+) cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2(+)Corin(+) cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.
Project description:Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.
Project description:The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.
Project description:Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1(-/-) mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1(-/-);Wnt5a(-/-) mice revealed a greater loss of Nurr1(+) cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson's disease.
Project description:Wingless-related MMTV integration site 1 (WNT1)/?-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/?-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/?-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1?) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/?-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.