use-freeze-thawed-embryos-for-high-efficiency-production-genetically

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

sarcomere-shortening-pluripotent-stem-cell-derived-cardiomyocytes

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Jichi Medical University

Hcn4, which encodes the hyperpolarization-activated, cyclic nucleotide-sensitive channel (I(h)), is a well-established marker of the cardiac sino-atrial node. We aimed to identify cis-elements in the genomic locus of the Hcn4 gene that regulate the transcription of Hcn4.

The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2.

Pigs with X-linked severe combined immunodeficiency (X-SCID) caused by a mutation of the interleukin-2 receptor gamma chain gene (IL2RG) are of value for a wide range of studies. However, they do not survive longer than 8 weeks because of their susceptibility to infections. To allow longer survival of X-SCID pigs, the animals must be born and reared under germ-free conditions. Here, we established an efficient system for piglet derivation by hysterectomy and used it to obtain and maintain a germ-free X-SCID pig. In four trials using pregnant wild-type pigs, 66% of piglets after hysterectomy started spontaneous breathing (range of 20-100% per litter). The resuscitation rate was found to negatively correlate with elapsed time from the uterus excision to piglet derivation (r=-0.97, P<0.05). Therefore, it is critical to deliver piglets within 5 min to achieve a high resuscitation rate (82% estimated from regression analysis). In a fifth trial with an IL2RG pig, four piglets were delivered within 4.2 min of uterus excision and three were alive (75%). One of the live born piglets was genotypically and phenotypically determined to be X-SCID and was reared for 12 weeks. The X-SCID piglet was free from both bacteria and fungi at all time points tested by microbial culture and grew without any abnormal signs or symptoms. This study showed successful production and rearing of germ-free pigs, enabling experiments involving long-term follow-up of X-SCID pigs.

Production and rearing of germ-free X-SCID pigs.

The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.

Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification.

The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging.

Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain.

X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7-nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

Generation of novel Il2rg-knockout mice with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas9.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP PSC-CMs exhibited more mature phenotypes than RFP cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.

A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation.

Cardiovascular diseases are the leading cause of death worldwide. Therefore, the discovery of induced pluripotent stem cells (iPSCs) and the subsequent generation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) was a pivotal point in regenerative medicine and cardiovascular research. They constituted an appealing tool for replacing dead and dysfunctional cardiac tissue, screening cardiac drugs and toxins, and studying inherited cardiac diseases. The problem is that these cells remain largely immature, and in order to utilize them, they must reach a functional degree of maturity. To attempt to mimic  environment, various methods including prolonging culture time, co-culture and modulations of chemical, electrical, mechanical culture conditions have been tried. In addition to that, changing the topology of the culture made huge progress with the introduction of the 3D culture that closely resembles the  cardiac topology and overcomes many of the limitations of the conventionally used 2D models. Nonetheless, 3D culture alone is not enough, and using a combination of these methods is being explored. In this review, we summarize the main differences between immature, fetal-like hiPSC-CMs and adult cardiomyocytes, then glance at the current approaches used to promote hiPSC-CMs maturation. In the second part, we focus on the evolving 3D culture model - it's structure, the effect on hiPSC-CMs maturation, incorporation with different maturation methods, limitations and future prospects.

A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes.

Transcriptome landscape of organs from mice and humans offers perspectives on the process of how organs develop and the similarity and diversity in each organ between the species. Among multi-species and multi-organ dataset, which was previously generated, we focused on the mouse and human dataset and performed a reanalysis to provide a more specific perspective on the maturation of human cardiomyocytes. First, we examined how organs diversify their transcriptome during development across and within two species. We unexpectedly identified that ribosomal genes were differentially expressed between mice and humans. Second, we examined the corresponding ages of organs in mice and humans and found that the corresponding developmental ages did not match throughout organs. Mouse hearts at P0-3 and human hearts at 18-19 wpc showed the most proximity in the regard of the transcriptome. Third, we identified a novel set of maturation marker genes that are more consistent between mice and humans. In contrast, conventionally used maturation marker genes only work well with mouse hearts. Finally, we compared human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) in maturation-enhanced conditions to human fetal and adult hearts and revealed that human PSC-CMs only expressed low levels of the potential maturation marker genes. Our findings provide a novel foundation to study cardiomyocyte maturation and highlight the importance of studying human samples rather than relying on a mouse time-series dataset.

Comparative Transcriptome Landscape of Mouse and Human Hearts.

We report that a sheep fetal liver provides a microenvironment for generating hematopoietic cells with long-term engrafting capacity and multilineage differentiation potential from human induced pluripotent stem cell (iPSC)-derived hemogenic endothelial cells (HEs). Despite the promise of iPSCs for making any cell types, generating hematopoietic stem and progenitor cells (HSPCs) is still a challenge. We hypothesized that the hematopoietic microenvironment, which exists in fetal liver but is lacking in vitro, turns iPSC-HEs into HSPCs. To test this, we transplanted CD45-negative iPSC-HEs into fetal sheep liver, in which HSPCs first grow. Within 2 months, the transplanted cells became CD45 positive and differentiated into multilineage blood cells in the fetal liver. Then, CD45-positive cells translocated to the bone marrow and were maintained there for 3 years with the capability of multilineage differentiation, indicating that hematopoietic cells with long-term engraftment potential were generated. Moreover, human hematopoietic cells were temporally enriched by xenogeneic donor-lymphocyte infusion into the sheep. This study could serve as a foundation to generate HSPCs from iPSCs.

Fetal sheep support the development of hematopoietic cells in vivo from human induced pluripotent stem cells.

We conducted two lines of genome-editing experiments of mouse hematopoietic stem cells (HSCs) with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). First, to evaluate the genome-editing efficiency in mouse bona fide HSCs, we knocked out integrin alpha 2b () with Cas9 ribonucleoprotein (Cas9/RNP) and performed serial transplantation in mice. The knockout efficiency was estimated at approximately 15%. Second, giving an example of X-linked severe combined immunodeficiency (X-SCID) as a target genetic disease, we showed a proof-of-concept of universal gene correction, allowing rescue of most of X-SCID mutations, in a completely non-viral setting. We inserted partial cDNA of interleukin-2 receptor gamma chain () into intron 1 of  via non-homologous end-joining (NHEJ) with Cas9/RNP and a homology-independent targeted integration (HITI)-based construct. Repaired HSCs reconstituted T lymphocytes and thymuses in SCID mice. Our results show that a non-viral genome-editing of HSCs with CRISPR/Cas9 will help cure genetic diseases.

ArticleTotal : 2	Year
Publication title Cited by 6	2020
Publication title Cited by 2	2021

Article	Year
The cardiac pacemaker-specific channel Hcn4 is a direct transcriptional target of MEF2. Cardiovascular research\| PubMed ID: 19477969	2009
Production and rearing of germ-free X-SCID pigs. Experimental animals\| PubMed ID: 29162766	2018
Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification. Cell stem cell\| PubMed ID: 30100166	2018
Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain. Journal of neuroscience methods\| PubMed ID: 30684509	2019
Generation of novel Il2rg-knockout mice with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas9. Experimental animals\| PubMed ID: 31801915	2019
A Novel Fluorescent Reporter System Identifies Laminin-511/521 as Potent Regulators of Cardiomyocyte Maturation. Scientific reports\| PubMed ID: 32144297	2020
A Brief Review of Current Maturation Methods for Human Induced Pluripotent Stem Cells-Derived Cardiomyocytes. Frontiers in cell and developmental biology\| PubMed ID: 32266260	2020
Comparative Transcriptome Landscape of Mouse and Human Hearts. Frontiers in cell and developmental biology\| PubMed ID: 32391358	2020
Fetal sheep support the development of hematopoietic cells in vivo from human induced pluripotent stem cells. Experimental hematology\| PubMed ID: 33395577	2021
Non-viral genome-editing in mouse bona fide hematopoietic stem cells with CRISPR/Cas9. Molecular therapy. Methods & clinical development\| PubMed ID: 33614821	2021

Hideki Uosaki

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