Evaluation of TH-Cre knock-in cell lines for detection and specific targeting of stem cell-derived dopaminergic neurons

The focal and progressive degeneration of dopaminergic (DA) neurons in ventral midbrain has made Parkinson's disease (PD) a particularly interesting target of cell-based therapies. However, ethical issues and limited tissue availability have so far hindered the widespread use of human fetal tissue in cell-replacement therapy. DA neurons derived from human pluripotent stem cells (hPSCs) offer unprecedented opportunities to access a renewable source of cells suitable for PD therapeutic applications. To better understand the development and functional properties of stem-cell derived DA neurons, we generated targeted hPSC lines with the gene coding for Cre recombinase knocked into the TH locus. When combined with flexed GFP, they serve as reporter cell lines able to identify and isolate TH+ neurons in vitro and after transplantation in vivo. These TH-Cre lines provide a valuable genetic tool to manipulate DA neurons useful for the design of more precise DA differentiation protocols and the study of these cells after transplantation in pre-clinical animal models of PD

Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurrl(+) stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery

Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson’s disease, STEM-PD

Cell replacement therapies for Parkinson’s disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022

Characterization of human dopaminergic neurons in the developing mesencephalon and upon differentiation of stem cells- for replacement therapy in Parkinson´s disease

Cell-replacement therapy is a promising approach for treating patients with Parkinson ́s disease (PD). For this purpose, there is a need for developing a protocol that can generate high numbers of human transplantable mesencephalic dopaminer- gic (mesDA) neurons. For decades, studies have therefore been made in mouse and other model organisms in order to elucidate key-factors that can be used for proper characterization and patterning of cells to become mesDA neurons. However, limited numbers of studies have been performed in human to confirm the expression and role of these factors. In this thesis, I have analysed the developing human mesencephalon for expression of key-fate determining proteins, which are known to be important in mesDA neuron development in the mouse. These key-factors were shown to exhibit a similar spatiotemporal expression pattern in the human brain, suggesting a conserved role for these proteins in mesDA neuron development across species. We were also able to confirm that human mesDA neurons are derived from radial glial cells in the floor plate (FP), positioned in the most ventral part of the mesencephalon. With the help of human specific mesDA markers we could optimize and develop a protocol that successfully patterns human embryonic stem cells (hESCs) into functional mesDA neuron progenitors. This protocol is one of the first to allow the generation of authen- tic mesDA neuron progenitors through a FP stage, mimicking early human mesDA neuron development. Furthermore, these cells survive transplantation and can restore motor deficits in a rat Parkinson ́s disease (PD) model. Thus, these cells show a prom- ising potential to be further developed for clinical use, treating patients with PD. In addition to patterning cells to a mesencephalic (midbrain) fate, we were also able to regionalize hESCs to neural progenitors resembling those of the human em- bryonic forebrain and hindbrain. This protocol, in combination with the generation of a SOX1-GFP hESC reporter cell line and the expression of the cell-surface marker CORIN by human FP cells, allowed us to isolate pure populations of regionalized neu- roepithelial and FP cells for deep sequencing. From this study, we identified several microRNAs with potential roles in the specification and development of human neural progenitors populations, including mesDA neuron progenitors. This opens up the possibility to further explore the mechanisms behind the specification of cells within the human central nervous system and can potentially be used for further development and optimization of protocols specifying human neural progenitor subtypes

Generating regionalized neuronal cells from pluripotency, a step-by-step protocol.

Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or in vitro analysis

Organization of the human embryonic ventral mesencephalon.

The neurons in the ventral mesencephalon (VM) are organized into several nuclei consisting of distinct neuronal populations. These include the dopaminergic (DA) neurons of the substania nigra and ventral tegmental area, the oculomotor (OM) neurons that innervate the muscles controlling eye movement, and the reticular neurons of the red nucleus (RN) involved in motor control and coordination reviewed in Puelles (2007). The factors and genes that control the differentiation of the various neuronal populations in the VM have been extensively studied in the mouse and other model organisms but little is known about the progenitors and their protein expression in the developing human brain. In this study we analyze if key regulators identified in rodents are also expressed in the human VM during embryonic development. We report that BLBP and LMX1A mark the floor plate and that FOXA2 is expressed in both the floor plate and basal plate of the human VM. The proneural transcription factors NGN2 and MASH1 are expressed in the ventricular zone of the human VM within and lateral to the floor plate. The post-mitotic DA neurons express TH as well as NURR1 and PITX3. ISL1 and BRN3A can be used to detect the cells of OM and RN, respectively. We show that many key developmental control factors are expressed in a temporal and spatial manner in the human VM essentially corresponding to what has been observed in the mouse. This data therefore suggest similar roles for these factors also in human VM development and dopamine neurogenesis

Human foetal brain tissue as quality control when developing stem cells towards cell replacement therapy for neurological diseases.

Human foetal brain tissue has been used in experimental and clinical trials to develop cell replacement therapy in neurodegenerative disorders such as Parkinson's disease and Huntington's disease. These pioneering clinical studies have shown proof of principle that cell replacement therapy can be effective and is worthwhile to develop as a therapeutic strategy for repairing the damaged brain. However, because of the limited availability of foetal brain material, and difficulties in producing standardized and quality-tested cell preparations from this source, there have been extensive efforts in investigating the potential use of alternative cell sources for generating a large number of transplantable, authentic neural progenitors and neurons. In this review, we highlight the value of using human foetal tissue as a reference material for quality control of acquired cell fate of in vitro generated neurons before and after transplantation

Vibration thresholds are increased at low frequencies in the sole of the foot in diabetes a novel multi-frequency approach.

AIMS: To evaluate multi-frequency tactilometry as a method to measure vibrotactile sense in the sole of the foot in subjects with diabetes. METHODS: Vibration thresholds were investigated at five frequencies (8, 16, 32, 64 and 125 Hz) at three sites (first and fifth metatarsal heads and heel) in the sole of the foot in subjects with Type 1 and Type 2 diabetes (n = 37). Thresholds were compared with healthy, age- and gender-matched subjects (n = 37) and related to glycaemic levels, subjective estimation of sensation in the feet and to perception of touch. RESULTS: Vibration thresholds were significantly higher in subjects with diabetes compared with healthy subjects at low frequencies (8, 16 and 32 Hz) at all measured sites, and also at 64 Hz for the metatarsal heads. Perception of touch and subjective estimation of sensation were significantly impaired in subjects with diabetes. Glycaemic levels, which were higher in subjects with diabetes, did not correlate with vibration thresholds at 32 Hz (most sensitive to Meissner's corpuscles) or with touch thresholds in subjects with diabetes. Vibration thresholds at 32 Hz correlated significantly with perception of touch (rho = 0.45-0.65; P < 0.01) and with subjective sensation (rho = -0.38 to -0.52; P < 0.001) in subjects with diabetes. Perception of touch and subjective estimation of sensation did also correlate (rho = -0.51 to -0.80; P < 0.002). CONCLUSIONS: Tactilometry is effective in detecting neuropathy in the sole of the foot at low frequencies of mainly 8-32 Hz, indicating that at least Meissner's corpuscles, or their related large nerve fibres, are affected by diabetes. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK

Dopamine neuron precursors within the developing human mesencephalon show radial glial characteristics.

Specification and differentiation of neural precursors into dopaminergic neurons within the ventral mesencephalon has been subject to much attention due to the implication of dopaminergic neurons in Parkinson's disease and the perspective of generating sources of therapeutically active cells to be used for cell replacement therapy for the disease. However, despite intensive research efforts, little is known about the characteristics of the dopamine neuron progenitors in human. We show that the dopamine neuron determinant LMX1a is expressed in the diencephalic and mesencephalic dopaminergic neuron domains during human development. Within the mesencephalon, LMX1a is expressed in the dopaminergic neurons and their progenitors located in the ventricular zone of the floor plate region. Furthermore, the neural progenitors in the developing human ventral mesencephalon have a radial morphology and express the radial glial markers Vimentin and BLBP. These radial glia are mitotic and act as precursors for the dopaminergic neurons. Finally, we show that progenitors isolated from the human ventral mesencephalon maintain their radial glial characteristics and neurogenic capacity after expansion in vitro, making them a promising future source of cells to be used in cell replacement therapy for Parkinson's disease. (c) 2009 Wiley-Liss, Inc

Generation of Regionally Specified Neural Progenitors and Functional Neurons from Human Embryonic Stem Cells under Defined Conditions

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease