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Unlocking embryonic stem cells
Scientists continually work to unlock the process by which embryonic stem cells (ESCs) grow into the many tissues of the body. Basic research wants to use stem cells in medical therapies, but the hurdle is stimulating ESCs to differentiate on command, so to speak, to be used for specific tissue repair. The human body has more than 200 different types of tissue cells. Each has the exact same DNA, but express genes differently in order to create tissue types with specific functions. Every one of the body's cells originates from embryonic stem cells as they are pluripotent or can become (differentiate) into any of our body's cell types.
A very active area of biological research is how to induce already differentiated cells to return to a pluripotent state. Using genetic and biochemical influences, scientists can reverse a differentiated cell - such as a skin fibroblast cell - backwards into its original pluripotent form. These new cells are called induced pluripotent stem cells or iPSCs.
Such iPSC cells have the potential to create tissue useful in regenerative medicine, such as in the repair of the heart following a heart attack, or in the creation of models of human diseases, and in making cells that enable drug screening tests before use with patients. But future progress with iPSCs needs more information on the basic biology behind pluripotency - what makes a cell differentiate?
"To use stem cells in therapy, the most important step is differentiation of iPSCs. We need to be able to differentiate iPSCs into a disease-relevant cell type with high efficiency and high purity."
In a study published in Stem Cell Reports, Zhao and colleagues have partly solved a long-unanswered basic question about stem cells: Why pluripotent stem cells with mutations blocking production of microRNAs are unable to differentiate?
They found those microRNA-deficient pluripotent stem cells need a specific element in order to differentiate into various neural cell types including neural cells with markers for: dopaminergic — midbrain neurons, the main source of dopamine (DA); glutamatergic — or glutamate, powerful excitatory neurotransmitters responsible for signalling between nerve cells, for learning and memory; and GABAergic neurons — GABA is the main inhibitory neurotransmitter in adult brains, and also important in learning and memory.
Neural cell differentiation in microRNA-deficient cells turns out to be simple: missing is a single microRNA producing a single protein.
In the Stem Cell Reports study, the group shows that a microRNA-302 mimic - delivered by a specially constructed lentivirus - was able to stimulate neural differentiation of mouse embryonic stem cells lacking Dgcr8, a vital gene needed to process the more than 2,000 microRNAs in cells. When they examined gene expression profiles in cells that do differentiate, they saw changes in sets of genes regulated by the protein p53. Also called tumor suppressor gene, p53 is known as "the guardian of the genome" for its many roles in preventing DNA damage and cancer.
Research shows that microRNA-302 reduces p53 being expressed in microRNA-deficient embryonic stem cells. It does this by binding to the 3' untranslated region of p53 mRNA.
They further show that direct inhibition of p53, or even deleting the gene itself, allowed embryonic stem cells and iPSCs to continue to become different cell types without the need for the microRNA-302. It appears the barrier preventing neurons from specializing is expression of p53.
• miR-302 enables neural differentiation of differentiation-incompetent Dgcr8-/- ESCs
• miR-302 directly suppresses p53 expression
• p53 inhibits neural differentiation of Dgcr8-/- and wild-type PSCs
• p53 may eliminate genetically defective embryos to save maternal resources
Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8-/- or Dicer-/– embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8-/- ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8-/- ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8-/- PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs.
Authors: Zhong Liu, Cheng Zhang, Maria Skamagki, Alireza Khodadadi-Jamayran, Wei Zhang, Dexin Kong, Chia-Wei Chang, Jingyang Feng, Xiaosi Han, Tim M. Townes, Hu Li, Kitai Kim, Rui Zhao.
Support came from a UAB startup fund, the UAB Faculty Development Fund, and UAB CFRC Pilot & Feasibility Grant ROWE15R0; National Institutes of Health grants HL093212, AG043531, CA196631-01A1 and NS095626; the TriStem-Star Foundation; Louis V. Gerstner Jr. Young Investigators award; Geoffrey Beene Junior Chair Award; Sidney Kimmel Scholar Award; Alfred W. Bressler Scholars Endowment Fund; MSKCC Society Fund; Paul F. Glenn Foundation; and the Mayo Clinic Center for Individualized Medicine.
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(A–C) Immunostaining of neuron-specific markers TUJ1 (green) and MAP2 (red) in embryoid bodies (EBs) formed by (A–A?) wild-type, (B–B?) Dgcr8-/--shctrl, and (C–C?) Dgcr8-/--302 ESCs.
Scale bars, 50 um. Image credit: The Author(s).