Marie E. Jönsson

Efficient production of mesencephalic dopamine neurons by Lmx1a expression in embryonic stem cells

Signaling factors involved in CNS development have been used to control the differentiation of embryonic stem cells (ESCs) into mesencephalic dopamine (mesDA) neurons, but tend to generate a limited yield of desired cell type. Here we show that forced expression of Lmx1a, a transcription factor functioning as a determinant of mesDA neurons during embryogenesis, effectively can promote the generation of mesDA neurons from mouse and human ESCs. Under permissive culture conditions, 75%–95% of mouse ESC-derived neurons express molecular and physiological properties characteristic of bona fide mesDA neurons. Similar to primary mesDA neurons, these cells integrate and innervate the striatum of 6-hydroxy dopamine lesioned neonatal rats. Thus, the enriched generation of functional mesDA neurons by forced expression of Lmx1a may be of future importance in cell replacement therapy of Parkinson disease.

The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease

Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson’s disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson’s disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson’s disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells.

Polyelectrolyte–macroion complexation. I. Effect of linear charge density, chain length, and macroion charge

The complexation between a linear flexible polyelectrolyte and one or several oppositely charged macroions was examined by employing a simple model system with focus on the electrostatic interactions. The composition and the structure of the complex as well as conformational data of the polyelectrolyte were obtained by using Monte Carlo simulations. These properties were investigated at different linear charge densities of the polyelectrolyte, different chain lengths of the polyelectrolyte, and different macroion charges, all at different numbers of macroions at constant volume. The binding isotherms obtained are Langmuir type, and in excess of macroions the polyelectrolyte–macroion complex displays a charge reversal. Upon complexation, the polyelectrolyte extension first reduces and thereafter increases as the number of complexed macroions increases, the minimal extension appearing for a neutral complex. Macroions prefer to complex to central polyelectrolyte segments, but for a neutral or an overcharged ...

Polyelectrolyte–macroion complexation. II. Effect of chain flexibility

A simple model with focus on the electrostatic interaction has been used to examine the complexation of a linear polyelectrolyte possessing variable flexibility with one or several oppositely charged macroions. Composition, structure, and thermodynamic properties of the complexes were obtained by using Monte Carlo simulations. Binding isotherms obtained were Langmuir-type with a quantitative binding up to a neutral complex with a plateau value corresponding to ∼50% overcharging, the largest overcharging appearing for the stiffest chain. Free energy calculations demonstrated that the complexation of the first macroion becomes less favorable as the chain stiffness is increased, whereas the opposite was found for a complexation of a macroion to a neutral complex. For a neutral complex and with a flexible chain, the repulsion between complexed macroions is strongly screened and the complexed macroions are located near each other. However, for a very stiff chain, the macroions are sequentially positioned along...

Identification of Transplantable Dopamine Neuron Precursors at Different Stages of Midbrain Neurogenesis.

Protocols used for generation of mesencephalic dopamine (mesDA) neurons from stem cells, or fetal brain tissue, invariably result in cell preparations that are highly mixed in composition, containing mesDA neuron precursors in various states of fate commitment and differentiation. For further optimisation and refinement of these procedures it is essential to determine the optimal stage of development and phenotypic characteristics of cells used for grafting. We have used fluorescence-activated cell sorting procedures to isolate mesDA precursors in defined stages of differentiation from mouse ventral mesencephalon (VM), at embryonic day 10.5 (E10.5), when the mesDA neuron domain consists of proliferative radial glia-like cells expressing the mesDA neuron determinant Lmx1a and the floorplate marker Corin, and at E12.5, when the VM has expanded to comprise a mixture of proliferative progenitors, neuroblasts and young neurons. The sorted cells were transplanted to the striatum of 6-hydroxydopamine-lesioned rats. Results show that the Lmx1a/Corin-expressing ventricular zone progenitors, which are the source of mesDA neurons in grafts from E10.5 VM, had lost this capacity at E12.5. At this later stage all transplantable mesDA precursors resided in the intermediate zone as postmitotic Nurr1-expressing neuroblasts. The more differentiated, TH-expressing cells survived sorting and transplantation poorly. We also provide evidence that, during early mesDA neurogenesis, the progenitors for nigral mesDA neurons segregate to lateral parts of the Lmx1a-expressing domain and can be selectively isolated based on their level of Corin expression. These results have implications for current efforts to develop well-characterized stem cell-derived mesDA progenitor cell preparations for cell therapy.

TRIM28 Represses Transcription of Endogenous Retroviruses in Neural Progenitor Cells

SUMMARY TRIM28 is a corepressor that mediates transcriptional silencing by establishing local heterochromatin. Here, we show that deletion of TRIM28 in neural progenitor cells (NPCs) results in high-level expression of two groups of endogenous retroviruses (ERVs): IAP1 and MMERVK10C. We find that NPCs use TRIM28-mediated histone modifications to dynamically regulate transcription and silencing of ERVs, which is in contrast to other somatic cell types using DNA methylation. We also show that derepression of ERVs influences transcriptional dynamics in NPCs through the activation of nearby genes and the expression of long noncoding RNAs. These findings demonstrate a unique dynamic transcriptional regulation of ERVs in NPCs. Our results warrant future studies on the role of ERVs in the healthy and diseased brain.

Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors

In this study, we have used a microRNA-regulated lentiviral reporter system to visualize and segregate differentiating neuronal cells in pluripotent cultures. Efficient suppression of transgene expression, specifically in undifferentiated pluripotent cells, was achieved by using a lentiviral vector expressing a fluorescent reporter gene regulated by microRNA-292. Using this strategy, it was possible to track progeny from murine ES, human ES cells, and induced pluripotent stem cells as they differentiated toward the neural lineage. In addition, this strategy was successfully used to FACS purify neuronal progenitors for molecular analysis and transplantation. FACS enrichment reduced tumor formation and increased survival of ES cell–derived neuronal progenitors after transplantation. The properties and versatility of the microRNA-regulated vectors allows broad use of these vectors in stem cell applications.

Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons.

Summary Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson’s disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models.

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 Parkinsons 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 Parkinsons disease.

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

Significance An important challenge for improving cell-based approaches for Parkinson’s disease is the development of techniques that facilitate greater standardization of the donor material. This report describes the enrichment of transplantable progenitors for dopamine neurons from the ventral mesencephalon based on targeting of transmembrane proteins. It is an important step toward the development of clinically relevant techniques that allow for greater standardization of cell preparations used in transplantation and potentially, more predictable clinical outcomes. The findings are highly relevant for current efforts to develop stem cell-based therapies for Parkinson’s disease, where current techniques yield mixed cell populations that may contain unwanted cell types and thus, would benefit from a cell selection step prior to grafting. An important challenge for the continued development of cell therapy for Parkinson’s disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a “FACS-array” approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.