Scientists have made breakthroughs in the study of transforming skin cells into neuronal cells

Scientists transform skin cells into neuronal cells

Dr. Zhiping collaborated with Dr. Ami Citri to make breakthrough research in the manipulation of human embryos and the transformation of postnatal fibroblasts into functional neuronal cells (iN).

application

- Single cell gene expression

Fluidigm technology

- Biomark system

- 48.48 Dynamic Microfluidic Integration Chip

Introduction

The Stanford University School of Medicine is known for transforming groundbreaking medical research to provide quality care to patients. Dr. Zhiping (postdoctoral fellow of the Department of Molecular and Cellular Physiology) and Dr. Ami Citri (postdoctoral fellow of the Department of Psychiatry and Behavior) combined their efforts with other researchers in the journal Nature Protocols entitled Induction of human neuronal cells by Supplemental transcription factors, Nature; and also Comprehensive qPCR profiling of gene expression in single neuronal cells. This article and other related procedures describe how they transform stem cells and postpartum skin cells into neural (brain) cells. Dr Pang said: "These work is not only an important step in the use of cell models to study human neurological diseases, but also an important reference for understanding how epigenetics regulates the differentiation and maturation of nerve cells."

challenge

The authors tested two hypotheses in this paper: 1) to determine whether a mandatory expression of a transcription factor can induce neuronal cell characteristics in human pluripotent stem cells, and 2) whether non-neuronal human fibroblasts can also be converted into neurons. Dr. Citri said: "Ziping came to me. He needs this project to assess the diversity of cell populations at the single-cell level." He said: "To assess the efficiency of conversion to nerve cells, the level of mature cells and the conversion The properties of specific nerve cells are fundamental to understanding the induction of neuronal cells from other cells. So we established a process to evaluate individual neurons using Fluidigm's dynamically integrated microfluidic chip. We are from a relatively large number of single cells. In the sample, a set of markers for glial biomarkers, neuronal markers, or fibroblasts was tested to assess the efficiency and specificity of the neuronal transformation process. We started with mouse neuronal cells and tried to detect the platform from Fluidigm. The quality of the data obtained, but its effectiveness immediately surprised us."

Dr Zhiping Pang is a postdoctoral fellow in the Department of Molecular and Cellular Physiology at Stanford University School of Medicine in writing this and the procedures mentioned in this report. He is currently an Assistant Professor in the Department of Neuroscience and Cell Biology at the New Jersey Children's Health Institute and Robert Wood Johnson Medical School. His work focuses on studying obesity.

Dr. Dr. Ami Citri conducted the study at the post-doctoral post of the Department of Psychiatry and Behavioral Sciences at Stanford University. In the summer of 2012, he will be a senior lecturer (Assistant Professor) at Hebrew University in Jerusalem. His work focuses on the study of addiction.

Dr Citri believes: "Fluidigm's method is very powerful, and I almost never go back to using traditional quantitative PCR methods."

solution

In their first experiment, in order to determine whether transcription factors in human pluripotent stem cells can induce neuronal properties, Brn2, ASCL1 and myt1l were transfected in undifferentiated human embryonic stem cells (ES) (author "BAM") with enhanced green fluorescent protein (EGFP). After treatment, they observed bipolar neuronal cells and mature neuronal morphology and expressed typical neuronal cells TUBB3 and MAP2. Electrophysiological analysis revealed that these cells produced action potentials. Therefore, BAM factors can induce differences in neural cells in human stem cells. In their second experiment, they wanted to know if non-neuronal human fibroblasts could also be converted directly into neurons. They transfected primary human fetal fibroblast cell lines (HFFs) with BAM factor + NEUROD1. These factors produce mature neuronal cells, express neurofilament proteins, and represent some of the neuronal characterization processes, including positive staining with synapsin and synaptotagmin (two synaptic vesicle proteins). Therefore, the BAM+NEUROD1 factor induces neuronal differentiation of non-neuronal human fibroblasts.

method

Single cell gene expression analysis was performed using Fluidigm's 48.48 dynamic chip. Single cells grown in the culture dish were collected by picking up the patch electrodes and ejected into CellsDirectTM buffer (Invitrogen), and then rapidly frozen until the next step. Thawed cells were subjected to specific target reverse transcription and 18 cycles of PCR for pre-amplification (STA). Preamplification products were subjected to real-time PCR analysis on a BioMark system. To ensure specific amplification, gradient titration of human brain total RNA was included in each experiment and only primers showing linear amplification were analyzed. In addition, PCR product melting curves for single cells and control RNA were compared to ensure the specificity of the PCR product. The BioMark system provides another level of data for validating immunofluorescence and electrophysiological phenotypic data. These researchers published their experimental journey in the journal Nature protocols: Comprehensive qPCR profiling of gene expression in single neuronal cells. Dr Pang said: "I have never been impressed when people talk about single-cell PCR in a traditional way. It's low-throughput, they can't run verification tests. But with Fluidigm's technology, we can With multiple internal controls, we can determine if an experiment works. The system gives us perfect results. What really surprised me is that when we repeated the experiment of one of the chips, we got almost the same result. So it is highly reproducible. We believe that others in the neuroscience community may be interested in doing this work. Therefore, we wrote an article about this operational process and accepted it by the magazine."

Technical advantages

Although these researchers mainly use the BioMark system for neuronal studies, they say they believe that other areas of research can also benefit from this technology. "Any study that requires the diversity of cell populations at the single cell level, and any areas that are limited by the starting materials, will be a place where Fuluda technology has a huge effect," Dr. Citri said. “This can be a biopsy of a person, or the first experiment from a small amount of tissue because the organ is too small, or it can be any microdissected sample that uses laser capture, or a very well defined cell population.”

Dr Pang said: "Single cell gene expression analysis is applicable in many fields, including cell biology and neurology. When heterogeneous cell populations, the recognition of cell-specific gene transcription becomes very important." After this study The two scientists have accepted academic positions in their new research institutions, but they all intend to continue using the Fuluda platform for single-cell research.

"Fluidigm's method is so powerful that I almost never go back to using traditional standard quantitative PCR methods," Dr. Citri said. “This is very useful because it allows me to validate microarray data in a highly efficient and cost-effective way. When I detect a large number of genes in various applications, I don’t need to use a microchip every time. Samples. I can use Furauda's microfluidic chip to study many samples with hundreds of probes. From the original microchip experiments I have created a database of candidate genes and quantitative PCR probes. In addition, the effective use of the smallest The amount of sample allows me to make my sample into a library, and I can continuously test them whenever a new set of genes becomes the focus of my interest. For me, this is a huge advantage of the Fluidigm system. one."

Dr Pang began working as an assistant professor at the New Jersey Children's Health Institute and the Department of Neuroscience and Cell Biology at Robert Wood Johnson Medical School in November 2011.

“My research focus is on studying the synaptic regulation mechanisms from stem cells to the brain. I am interested in understanding how obese people's food intake behavior is altered by the body's hormones and neuropeptides.” He said, “An interesting The phenomenon is that there is a group of neurons in the hypothalamic region that express leptin receptors, but their responses to leptin are very different. I conclude that different cells in the hypothalamus have different gene expression patterns, which can only be used. Single-cell gene expression profiling. My other research direction is to induce neuroblasts using human fibroblasts or pluripotent stem cells, using this as a cellular model to study human neurological diseases. Single-cell genetic analysis will be One of the main techniques used in these synaptic regulation studies in my laboratory."

“Any study that requires the diversity of cell populations at the single cell level, and any areas that are limited by the starting materials, will be a place where Fuluda technology has a huge effect.”

— Dr. Citri

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