Alzheimer’s disease: researchers succeeded in producing dopaminergic A9 neurons from human-induced pluripotent stem cells


A new study led by a researcher in the Jacobs School of Medicine and Biomedical Sciences at the University at Buffalo has important implications for developing future treatments for Parkinson’s disease (PD), a progressive nervous system disorder that affects movement and often includes tremors.

“In this study, we find a method to differentiate human induced pluripotent stem cells (iPSCs) to A9 dopamine neurons (A9 DA), which are lost in Parkinson’s disease,” says Jian Feng, Ph.D., professor of physiology and biophysics in the Jacobs School and the senior author on the paper published May 24 in Molecular Psychiatry.

“These neurons are pacemakers that continuously fire action potentials regardless of excitatory inputs from other neurons,” he adds. “Their pacemaking property is very important to their function and underlies their vulnerability in Parkinson’s disease.”

“This exciting breakthrough is a critical step forward in efforts to better understand Parkinson’s disease and how to treat it,” says Allison Brashear, MD, UB’s vice president for health sciences and dean of the Jacobs School. “Jian Feng and his team are to be commended for their innovation and resolve.”

Loss of neurons causes Parkinson’s movement symptoms

Feng explains there are many different types of dopamine neurons in the human brain, and each type is responsible for different brain functions.

Nigral dopamine neurons, also known as the A9 DA neurons, are responsible for controlling voluntary movements. The loss of these neurons causes the movement symptoms of Parkinson’s disease, he says.

“Scientists have been trying hard to generate these neurons from human pluripotent stem cells to study Parkinson’s disease and develop better therapies,” Feng says. “We have succeeded in making A9 dopamine neurons from human induced pluripotent stem cells. It means that we can now generate these neurons from any PD patients to study their disease.”

Feng notes that A9 DA neurons are probably the largest cells in the human body. Their volume is about four times the volume of a mature human egg.

“Over 99 percent of the volume is contributed by their extremely extensive axon branches. The total length of axon branches of a single A9 DA neuron is about 4.5 meters,” he says. “The cell is like the water supply system in a city, with a relatively small plant and hundreds of miles of water pipes going to each building.”

Quest to develop better treatment therapies

In addition to their unique morphology, the A9 DA neurons are pacemakers—they fire action potentials continuously regardless of synaptic input.

“They depend on Ca2+ channels to maintain the pacemaking activities. Thus, the cells need to deal with a lot of stress from handling Ca2+ and dopamine,” Feng says. “These unique features of A9 DA neurons make them vulnerable. Lots of efforts are being directed at understanding these vulnerabilities, with the hope of finding a way to arrest or prevent their loss in Parkinson’s disease.”

“Pacemaking is an important feature and vulnerability of A9 DA neurons. Now that we can generate A9 DA pacemakers from any patient, it is possible to use these neurons to screen for compounds that may protect their loss in PD,” Feng notes. “It is also possible to test whether these cells are a better candidate for transplantation therapy of PD.”

To differentiate human iPSCs to A9 DA neurons, the researchers tried to mimic what happens in embryonic development, in which the cells secrete proteins called morphogens to signal to each other their correct position and destiny in the embryo.

Feng notes the A9 DA neurons are in the ventral part of the midbrain in development.

“Thus, we differentiate the human iPSCs in three stages, each with different chemicals to mimic the developmental process,” he says. “The challenge is to identify the correct concentration, duration, and treatment window of each chemical.”

“The combination of this painstaking work, which is based on previous work by many others in the field, makes it possible for us to generate A9 DA neurons,” Feng adds.

Feng points out there are a number of roadblocks to studying Parkinson’s disease, but that significant progress is being made.

“There is no objective diagnostic test of Parkinson’s disease, and when PD is diagnosed by clinical symptoms, it is already too late. The loss of nigral DA neurons has already been going on for at least a decade,” he says. “There was previously no way to make human dopamine neurons from a PD patient so we could study these neurons to find out what goes wrong.”

Scientists have been using animal models and human cell lines to study Parkinson’s disease, but these systems are inadequate in their ability to reflect the situation in human nigral DA neurons, Feng says. “Just within the past 15 years, PD research has been transformed by the ability to make patient-specific dopamine neurons that are increasingly similar to their counterparts in the brain of a PD patient.”

Dopamine (DA) neurons in the ventral midbrain (VM) constitute a heterogeneous group of cells with different anatomical locations, physiological properties, and projection patterns. These cells are involved in a broad spectrum of cerebral functions associated with voluntary movement, as well as with cognitive and emotive tasks [1,2].

DA neurons are traditionally divided into three subtypes. A9 neurons are located in the substantia nigra pars compacta (SNpc), A10 in the ventral tegmental (VTA) area, and A8 in the retrorubral field. As well as displaying different functions and axonal innervations, they exhibit heterogeneous susceptibility to disease processes and show fundamental differences in vulnerability to cell death in Parkinson’s disease (PD). Each cell group forms specific connections and projects to distinct anatomical target areas of the central nervous system (CNS), establishing separately controlled functional networks [3,4,5]. A8 and A10 neurons innervate the ventral striatum, nucleus accumbens, septum, and the prefrontal cortex via the mesolimbic pathway, and are mainly involved in controlling emotional behavior and motivation. A9 neurons project to the striatum, forming the nigrostriatal pathway that regulates motor function. They also have different functional and projection patterns, as compared to the other subtypes. A9 neurons in humans and other primates also show accumulation of the neuromelanin pigment. A9 neurons are primarily degenerated in PD, making them the subject of more extensive studies [6,7,8,9,10].

Given the key role of A9 cells in PD, the generation of this subtype of DA neurons from stem cell sources is an area of intense investigation ultimately aimed at exploiting their use in cell-based replacement treatment. For decades, neuroscientists have attempted to identify selective markers expressed in these subpopulations, in order to dissect the complexity of DA regulatory networks and design more effective therapeutic strategies [11,12].

The inaccessibility of fetal and adult human brain tissue makes it difficult to elucidate the relation between histological assessments and the heterogeneity of DA neurons at the molecular level. The compilation of a comprehensive dataset linking the molecular diversity of DA neurons with their function and anatomical innervation target would require a systematic genome-wide molecular classification at single-cell resolution [13,14].

The advent of single cell sequencing technologies has provided unprecedented insights into DA subtypes and uncovered an unexpectedly high heterogeneity, even within anatomically defined DA subgroups. Such approaches have already been used in the adult mouse brain to unbiasedly catalog DA neurons, based on their gene expression profiles.

However, the question of whether a similar diversity exists in the human midbrain and whether molecularly distinct DA subtypes correspond to innervation target regions and the traditional classification based on anatomical landmarks, remains completely unexplored [3,15,16].

The ability to recreate human neurons from human pluripotent stem cells (hPSCs) opens up exciting opportunities to study human neurogenesis and understand mechanisms and treatments for brain disease(s). It also provides access to a renewable source of cells potentially suitable for therapeutic applications, including drug screening and cell-based therapy (Figure 1) [17,18].

The generation of human tissues in vitro, combined with the use of human cells in xenograft transplantation models and sophisticated transcriptomics technologies, is ushering in the new era of “human” biology. Breaking down the intricate regulatory system controlling DA neuron subtype specification into its individual layers will provide crucial new insights into the transcription factors and molecular cues that specifically drive the diversity of human DA neurons [19,20,21].

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Figure 1
Schematic illustration of potential DA progenitor sources for modeling VM differentiation and for therapeutic application in PD, including blastocysts and their pluripotent stem cell derivative (top-left), fetal midbrain tissue (bottom left), and somatic cells from adult individuals, such as a skin biopsy used to reprogram cells to pluripotency (bottom-right).

reference link :

More information: Hong Li et al, Generation of human A9 dopaminergic pacemakers from induced pluripotent stem cells, Molecular Psychiatry (2022). DOI: 10.1038/s41380-022-01628-1


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