Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.

CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed.

Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one.

[Establishment of Integration-Free Human Induced Pluripotent Stem Cells from A Patient with Primary Myelofibrosis].

Objective: To establish the primary myelofibrosis (PMF)-induced pluripotent stem cell line (iPSC) by means of iPSC techinique so as to provide a experimental model for studying the blood disease mechanisms.

Methods: Induced pluripotent stem cells were generated from mononuclear cells isolated from a PMF patient with JAK2(V617F) mutation by using episomal vectors.

Results: PMF-derived iPSC was established from the patient with JAK2(V617F) gene mutation.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging.

Growth of human umbilical cord Wharton's Jelly-derived mesenchymal stem cells on the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate).

As a new member of the polyhydroxyalkanoate (PHA) family, the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) was evaluated for its biocompatibility for human umbilical cord Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs). More WJ-MSC adhesion and proliferation were observed on PHBVHHx film compared with films made of poly(L-lactic acid) (PLA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Higher DNA synthesis by WJ-MSCs was detected on PHBVHHx film than on PLA, PHBV and PHBHHx films.