Single-cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.

Stem cell technologies provide new opportunities for modeling cells in health and disease and for regenerative medicine. In both cases, developmental knowledge and defining the molecular properties and quality of the cell types is essential. In this study, we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into functional midbrain dopaminergic (mDA) neurons. We found that laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3ß inhibition plus FGF8b, improved midbrain patterning. In addition, neurogenesis and differentiation were enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high-quality molecularly defined midbrain cell types, including mDA neurons. Our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly defined hESC-derived cell types in Parkinson disease.

Transcriptional synergy as an emergent property defining cell subpopulation identity enables population shift.

Single-cell RNA sequencing allows defining molecularly distinct cell subpopulations. However, the identification of specific sets of transcription factors (TFs) that define the identity of these subpopulations remains a challenge. Here we propose that subpopulation identity emerges from the synergistic activity of multiple TFs. Based on this concept, we develop a computational platform (TransSyn) for identifying synergistic transcriptional cores that determine cell subpopulation identities. TransSyn leverages single-cell RNA-seq data, and performs a dynamic search for an optimal synergistic transcriptional core using an information theoretic measure of synergy. A large-scale TransSyn analysis identifies transcriptional cores for 186 subpopulations, and predicts identity conversion TFs between 3786 pairs of cell subpopulations. Finally, TransSyn predictions enable experimental conversion of human hindbrain neuroepithelial cells into medial floor plate midbrain progenitors, capable of rapidly differentiating into dopaminergic neurons. Thus, TransSyn can facilitate designing strategies for conversion of cell subpopulation identities with potential applications in regenerative medicine.

Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP.

Parkinson's disease (PD) is a neurodegenerative disorder in which the loss of dopaminergic neurons in the midbrain (mDA neurons) causes progressive loss of motor control and function. Using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells, we investigated the mechanisms controlling the survival of mDA neurons. We found that the extracellular matrix protein laminin-511 (LM511) promoted the survival and differentiation of mDA neurons. LM511 bound to integrin α3ß1 and activated the transcriptional cofactor YAP. LM511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death-associated protein PTEN. In addition, LM511-YAP signaling increased the expression of transcription factors critical for mDA identity, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess therapeutic potential for PD patients. We propose that by enhancing LM511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies.

Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells.

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.

Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors.

Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson disease. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. However, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells after cessation of normal dopamine neurogenesis. We show here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone beyond the major dopamine neurogenic period. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can be stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors as well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis.

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells.

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.

Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis.

Liver X receptors (Lxrα and Lxrß) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host.

How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

Control of neural stem cell adhesion and density by an electronic polymer surface switch.

Adhesion is an essential parameter for stem cells. It regulates the overall cell density along the carrying surface, which further dictates the differentiation scheme of stem cells toward a more matured and specified population as well as tissue. Electronic control of the seeding density of neural stem cells (c17.2) is here reported. Thin electrode films of poly(3,4-ethylenedioxythiophene) (PEDOT):Tosylate were manufactured along the floor of cell growth dishes. As the oxidation state of the conjugated polymer electrodes was controlled, the seeding density could be varied by a factor of 2. Along the oxidized PEDOT:Tosylate-electrodes, a relatively lower density of, and less tightly bonded, human serum albumin (HSA) was observed as compared to reduced electrodes. We found that this favors adhesion of the specific stem cells studied. Surface analysis experiments, such as photoelectron spectroscopy, and water contact angle measurements, were carried out to investigate the mechanisms responsible for the electronic control of the seeding density of the c17.2 neural stem cells. Further, our findings may provide an opening for electronic control of stem cell differentiation.

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice.

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: an efficient platform for cell adhesion.

Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications.

Requirement for thyroid hormone receptor beta in T3 regulation of cholesterol metabolism in mice.

T3 potently influences cholesterol metabolism through the nuclear thyroid hormone receptor beta (TRbeta), the most abundant TR isoform in rodent liver. Here, we have tested if TRalpha1, when expressed at increased levels from its normal locus, can replace TRbeta in regulation of cholesterol metabolism. By the use of TRalpha2-/-beta-/- animals that overexpress hepatic TRalpha1 6-fold, a near normalization of the total amount of T3 binding receptors was achieved. These mice are similar to TRbeta-/- and TRalpha1-/-beta-/- mice in that they fail to regulate cholesterol 7alpha-hydroxylase expression properly, and that their serum cholesterol levels are unaffected by T3. Thus, hepatic overexpression of TRalpha1 cannot substitute for absence of TRbeta, suggesting that the TRbeta gene has a unique role in T3 regulation of cholesterol metabolism in mice. However, examination of T3 regulation of hepatic target genes revealed that dependence on TRbeta is not general: T3 regulation of type I iodothyronine deiodinase and the low density lipoprotein receptor were partially rescued by TRalpha1 overexpression. These in vivo data show that TRbeta is necessary for the effects of T3 on cholesterol metabolism. That TRalpha1 only in some instances can substitute for TRbeta indicates that T3 regulation of physiological and molecular processes in the liver occurs in an isoform-specific fashion.