The genetic reprogramming technology allows one to generate pluripotent stem
cells for individual patients. These cells, called induced pluripotent stem
cells (iPSCs), can be an unlimited source of specialized cell types for the
body. Thus, autologous somatic cell replacement therapy becomes possible, as
well as the generation of in vitro cell models for studying
the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral
sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss
of upper and lower motor neurons. About 10% of cases are genetically inherited,
and the most common familial form of ALS is associated with mutations in the
SOD1 gene. We used the reprogramming technology to generate
induced pluripotent stem cells with patients with familial ALS.
Patient-specific iPS cells were obtained by both integration and transgene-free
delivery methods of reprogramming transcription factors. These iPS cells have
the properties of pluripotent cells and are capable of direct differentiation
into motor neurons.
Cytogenetic analysis of karyotypes of hESM01-hESM04 human embryonic stem cells and substrains derived from these strains showed that all these strains retained normal karyotype during long-term culturing. Two substrains of embryonic stem cells with chromosome aberrations indicating clonal origin of these strains were detected. The potentialities of using analysis of chromosome variability of embryonic stem cells for evaluation of predisposition of the corresponding genotypes to the formation of chromosome abnormalities are discussed.
Induced pluripotent stem cells (iPSCs) have the capacity to unlimitedly
proliferate and differentiate into all types of somatic cells. This capacity
makes them a valuable source of cells for research and clinical use. However,
the type of cells to be reprogrammed, the selection of clones, and the various
genetic manipulations during reprogramming may have an impact both on the
properties of iPSCs and their differentiated derivatives. To assess this
influence, we used isogenic lines of iPSCs obtained by reprogramming of three
types of somatic cells differentiated from human embryonic stem cells. We
showed that technical manipulations in vitro, such as cell
sorting and selection of clones, did not lead to the bottleneck effect, and
that isogenic iPSCs derived from different types of somatic cells did not
differ in their ability to differentiate into the hematopoietic and neural
directions. Thus, the type of somatic cells used for the generation of fully
reprogrammed iPSCs is not important for the practical and scientific
application of iPSCs.
DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau 1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.
Due to possible proliferative effects of karyotypic reorganization of human embryonic stem cell (hESC) lines detailed genetic analysis are indicated prior to any application of hESCs. Molecular cytogenetic analysis of two different hESC sublines was performed and revealed aberrant chromosomes in both of them, i.e. in hESM01r18 (46,ХХ,-18,+mar) and hESM0309 (46,ХХ,del(4),dup (9)). This study shows that microdissection and multicolor fl uorescence in situ hybridization (mFISH) can be used to detect the chromosomal changes precisely of the derivative chromosomes that are diffi cult to identify by conventional G-banded chromosome analysis. In the present study chromosome microdissection and reverse FISH were applied using multicolor fl uorescence in situ hybridization (mFISH) for detailed characterization of the derivative chromosomes. The karyotypes of hESC lines were described as: 46,ХХ,r(18)(::p11.31→q21.2::q21.2→p11.31::) and 46,XX,del(4)(q25q31.1),dup(9) (q12q33), respectively. The potential role of the chromosomal regions involved in rearrangements for cell proliferation is discussed.
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