Maintenance and differentiation Protocols | Embryonic stem cell | Stem Cell

An Efficient Method for Immortalizing Mouse Embryonic Fibroblasts by CRISPR-mediated Deletion of the Tp53 Gene

SS Srisathya Srinivasan

HH Hsin-Yi Henry Ho

2962 Views

Jan 20, 2025

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inconsistent or alter the physiological properties of the cells. Here, we describe an optimized method that overcomes these limitations. By using electroporation to deliver CRISPR constructs that target the Tp53 gene, the method reliably generates immortalized MEFs (iMEFs) within three weeks. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted broadly to immortalize other mouse primary cell types.

Generation of Mouse Primitive Endoderm Stem Cells

YO Yasuhide Ohinata

AS Atsunori Saraya

HK Haruhiko Koseki

0 Q&A

2583 Views

Nov 20, 2023

The blastocysts consist of dozens of cells of three distinct lineages: epiblast (Epi), trophoblast (TB), and primitive endoderm (PrE). All embryonic and extraembryonic tissues are derived from Epi, TB, and PrE. Stem cell lines representing preimplantation Epi and TB have been established and are known as embryonic stem cells (ESCs) and trophoblast stem cells (TSCs). Extraembryonic endoderm cells (XENCs) constitute a cell line that has been established from PrE. Although in vivo, PrE gives rise to visceral endoderm (VE), parietal endoderm (PE), and marginal zone endoderm (MZE); XENCs, on blastocyst injection into chimeras, primarily contribute to the distal region of PE. Here, we provide a comprehensive protocol for the establishment of fully potent primitive endoderm stem cell (PrESC) lines. PrESCs are established and maintained on mouse embryonic fibroblast (MEF) feeder cells in a serum-free medium supplemented with fibroblast growth factor 4 (FGF4), heparin, CHIR99021, and platelet-derived growth factor-AA (PDGF-AA). PrESCs co-express markers indicative of pluripotency and endoderm lineage commitment, exhibiting characteristics akin to those of PrE. On transplantation of PrESCs into blastocysts, they demonstrate a high efficiency in contributing to VE, PE, and MZE. PrESCs serve as a valuable model for studying PrE, sharing similarities in gene expression profiles and differentiation potential. PrESCs constitute a pivotal cornerstone for in vitro analysis of early developmental mechanisms and for studies of embryo reconstitution in vitro, particularly in conjunction with ESCs and TSCs.

Key features

• Establishment and maintenance of primitive endoderm stem cell (PrESCs) capable of recapitulating the developmental prowess inherent to PrE.

• Offering a source of PrE lineage for embryo-like organoid reconstitution studies.

Graphical overview

Isolation of Neural Stem Cells from the Embryonic Mouse Hippocampus for in vitro Growth or Engraftment into a Host Tissue

Oksana Rybachuk

Olga Kopach

TP Tetyana Pivneva

Vitaliy Kyryk

0 Q&A

10240 Views

Feb 20, 2019

For both stem cell research and treatment of the central nervous system disorders, neural stem/progenitor cells (NSPCs) represent an important breakthrough tool. In the expanded stem cell-based therapy use, NSPCs not only provide a powerful cell source for neural cell replacement but a useful model for developmental biology research. Despite numerous approaches were described for isolation of NSPCs from either fetal or adult brain, the main issue remains in extending cell survival following isolation. Here we provide a simple and affordable protocol for making viable NSPCs from the fetal mouse hippocampi, which are capable of maintaining the high viability in a 2D monolayer cell culture or generating 3D neuro-spheroids of cell aggregates. Further, we describe the detailed method for engraftment of embryonic NSPCs onto a host hippocampal tissue for promoting multilinear cell differentiation and maturation within endogenous environment. Our experimental data demonstrate that embryonic NSPCs isolated using this approach show the high viability (above 88%). Within a host tissue, these cells were capable of differentiating to the main neural subpopulations (principal neurons, oligodendrocytes, astroglia). Finally, NSPC-derived neurons demonstrated matured functional properties (electrophysiological activity), becoming functionally integrated into the host hippocampal circuits within a couple of weeks after engraftment.

Reprogram Murine Epiblast Stem Cells by Epigenetic Inhibitors

HZ Hui Zhang

YD Yali Dou

0 Q&A

7536 Views

Mar 5, 2017

Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 methylation by MLL1/KMT2A.

Differentiation of Human Embryonic Stem Cells into Cone Photoreceptors

Anthony Flamier

Andrea Barabino

Gilbert Bernier

1 Q&A

11275 Views

Jul 20, 2016

Photoreceptors are specialized retinal neurons able to respond to light in order to generate visual information. Among photoreceptors, cones are involved in colors discrimination and high-resolution central vision and are selectively depleted in macular degenerations and cone dystrophies. A possible therapeutic solution for these disorders is to replace degenerating cells with functional cones. Here, we describe a simple protocol for the rapid production of large amount of cone photoreceptors from human pluripotent stem cells. The differentiation protocol is based on the “default pathway” of neural induction using the BMP, TGFβ and WNT antagonist COCO.

Retinal Differentiation of Mouse Embryonic Stem Cells

Anna La Torre

5 Q&A

15960 Views

Jul 5, 2016

Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and G4 cell lines) and mouse induced-Pluripotent Stem Cell (iPSCs) lines.

Transfection of Embryoid Bodies with miRNA Precursors to Induce Cardiac Differentiation

Federica Pisano

MG Massimiliano Gnecchi

1 Q&A

9581 Views

Feb 5, 2016

In recent years, the utilization of stem cell therapy to regenerate cardiac tissue has been proposed as a possible strategy to treat cardiac damage (Gnecchi et al., 2012, Aguirre et al., 2013; Sanganalmath and Bolli, 2013). Although encouraging results have been obtained in experimental models, the efficiency of cardiac regeneration is very poor and one of the major barriers to progress in the area of cell therapy for damaged heart is represented by the limited capacity of cells to differentiate into mature cardiomyocytes (CMC) (Laflamme and Murry, 2011). Cell manipulation and transfection represent versatile tools in this context (Melo et al., 2005; Dzau et al., 2005). Murine P19 embryonal carcinoma cells are a well-established cell line capable of differentiating in vitro into spontaneously beating CMC. This cell system with its limited cell culture requirements, protocol reproducibility and ease in uptake and subsequent expression of ectopic genetic materials render it ideal for the study of the cardiac differentiation process. P19 cells have been successfully used to gain important insights into the early molecular processes of CMC differentiation (van der Heyden and Defize, 2003; van der Heyden et al., 2003). P19 cells can also be maintained in an undifferentiated state in a monolayer culture when grown in adherence; this condition allows the enrichment of large cell numbers useful for cardiac differentiation protocols (McBurney, 1993). On the other hand, when cultured in bacterial dishes, P19 cells will grow in suspension and generate embryoid bodies (EB). When exposed to dimethyl sulfoxide (DMSO), EB differentiate into spontaneously beating cells, which can be defined as CMC. This definition is based on their gene and protein expression and their electrophysiological properties (Wobus et al., 1994; van der Heyden et al., 2003). In our laboratory, we used this in vitro model to verify whether the over-expression of a defined combination of miRNA can synergistically induce effective cardiac differentiation (Pisano et al., 2015). We used miRNA1, miRNA133 and miRNA499 alone or in combination. Here, we describe how we transiently transfect P19 cells to over-express a single or a combination of miRNA precursors (pre-miRNA).

Wnt Reporter Activity Assay

Chen Zhao

2 Q&A

36744 Views

Jul 20, 2014

This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.

Hedgehog (Hh) Reporter Activity Assay

Chen Zhao

0 Q&A

15857 Views

Jul 20, 2014

This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.

Intestinal Differentiation of Mouse ESCs

SO Soichiro Ogaki

SK Shoen Kume

0 Q&A

8643 Views

Dec 20, 2013

ES cells (ESCs) are pluripotent and offer a good tool to study early embryonic development. Intestinal cells are derived from the definitive endoderm. In contrast to adult tissue stem cells, embryonic development and differentiation from ES cells have not been as well investigated in the intestine. There are four differentiated cell types of non-proliferative epithelial cells: enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Intestinal stem cells (ISCs) and progenitor cells reside in crypts, proliferate vigorously, and function as the source of differentiated epithelial cells. Here, we describe a protocol, in which differentiated cell types of the intestine are derived from mouse ESCs. In this protocol, we describe a protocol to differentiate mouse ES cells into Cdx2-expressing intestinal endoderm efficiently by co-culturing with M15, a mouse mesonephric cell line, and treatment with two chemical compounds. The chemical compounds used are BIO and DAPT. BIO is a Gsk3 inhibitor, that activate Wnt signaling pathway, and DAPT is a-secretase inhibitor that inhibit Notch signaling pathway. BIO and DAPT treatment yielded all representative cell lineages, enterocytes, goblet cells, enteroendocrine cells and paneth cells, to be derived from mouse ESCs.