Vol 19, No 4 (2024)

Mature hyaline cartilage has a low regenerative potential and its repair remains a complex clinical and research issue. Articular cartilage injuries often contribute to the development of osteoarthritis, resulting in loss of joint function and patient disability. Surgical techniques for repairing articular surfaces, such as mosaic chondroplasty and microfracture, which are designed for small defects, cannot be used for degenerative and dystrophic cartilage lesions. Cell therapy using chondrocytes differentiated from induced pluripotent stem cells (iPSCs) is a promising approach to reconstruct articular cartilage tissue. iPSCs have high proliferative activity, which allows the harvesting of autologous cells in quantities necessary to repair a joint defect. CRISPR-Cas genome editing technology, based on the bacterial adaptive immune system, enables the genetic modification of iPSCs to obtain progenitor cells with specific characteristics and properties.

This review describes specific research papers on the combined use of iPSC and CRISPR-Cas technologies for the evaluation of cartilage regenerative medicine. Papers were evaluated for the last twelve years since CRISPR-Cas technology was introduced to the global community. CRISPR-Cas is currently being used to address therapeutic issues in articular cartilage regeneration by increasing the efficiency of chondrogenic differentiation of iPSC lines and harvesting a more homogeneous population of chondroprogenitor cells. Another approach is to remove the pro-inflammatory cytokine receptor sequence to produce inflammation-resistant cartilage. Finally, knocking out genes for components of the major histocompatibility complex allows harvesting chondrocytes that are invisible to the recipient's immune system. This kind of research contributes to personalized healthcare and can improve the quality of life of the world's population in the long term.

BACKGROUND: Mammalian cell cultures play a key role in the pharmaceutical industry, requiring constant monitoring of the cell conditions during fermentation. In addition to monitoring of the physical-chemical parameters, it is important to evaluate the transcription state of cells, for which gene expression analysis is used. Currently, quantitative reverse transcription polymerase chain reaction (qPCR) is the dominant method. However, the loop-mediated isothermal amplification (LAMP) technique also attracts attention due to its high specificity, sensitivity and reaction rate. LAMP is becoming a promising tool for rapid analysis of gene expression, especially under the conditions of limited biological material or a large volume of samples.

AIM: Development of a technique for semi-quantifying the expression level of target genes in human cell culture Expi293 using LAMP.

MATERIALS AND METHODS: For LAMP, a recombinant large fragment of Bacillus stearothermophilus DNA polymerase (Bst-pol) was obtained, purified, and optimal reaction conditions were determined. SYBR Green I and LUCS13 were used as intercalating dyes. The amplification parameters for different concentrations of the enzyme and the dye were analyzed. Standard SYBR Green I kits were used for qPCR. Both methods were compared when analyzing the expression of IGF1, FGF2 and EIF3i genes in cell lines with an increased expression level of these genes.

RESULTS: It has been shown that using LUCS13 dye provides the classic S-shape of the signal accumulation curve in LAMP, while using SYBR Green I dye causes artifacts. The optimal concentration of Bst-pol was 40 ng/µl. When comparing the two methods, it was found that LAMP has greater sensitivity, allows determining gene expression with an accuracy comparable to qPCR, demonstrating a shorter reaction time (up to 35 minutes).

CONCLUSION: Although qPCR remains the main method for assessing the level of gene expression, LAMP offers a number of advantages that make it an attractive alternative for various biotechnological purposes. Due to its high speed, ease of execution and accessibility, as well as high sensitivity and specificity, LAMP is a valuable technique for rapid analysis of gene expression during cell culture monitoring.

BACKGROUND: Cystic fibrosis (CF) is the most common fatal and incurable genetic disease. The most frequent reason is the F508del mutation in the CFTR gene, which can theoretically be corrected by genome editing. Currently, pressing issue is the search for the most effective CRISPR/Cas9-based editing system for this mutation, as well as the selection of target cells that could support self-renewal and provide differentiated progeny of lung cells. Such promising targets can be presented by human induced pluripotent stem cells (hiPSCs) and human induced airway basal stem cells (hiBCs).

AIM: To correct the F508del mutation in the CFTR gene in hiPSCs and hiBCs derived from CF patients using CRISPR/Cas9 technology.

MATERIALS AND METHODS: We obtained three hiPSCs lines by reprogramming fibroblasts from patients with CF and three hiBCs lines by targeted differentiation of these hiPSCs. Based on three different variants of the Cas9 nuclease, three single guide RNAs, and two single-stranded oligodeoxyribonucleotides we created eight systems for editing the F508del mutation and transfected them into hiPSCs and hiBCs by electroporation. Then we analyzed the levels of non-homologous end joining, indels, and directed homologous repair in the transfected cells by deep target sequencing.

RESULTS: Eight editing systems were tested on hiPSCs. The highest efficiency of non-homologous end joining, indels, and directed homologous repair was observed when using SpCas9(1.1). The mutation was significantly corrected using a combination of this nuclease with the guide RNA sgCFTR#sp1 (with an efficiency up to 6.6% of alleles in transfected cells). Only two editing systems), which seemed to be most effective on chiPSCs, were tested on hiBCs. The mutation was significantly corrected using both systems (with efficiency up to 20% of alleles in transfected cells).

CONCLUSION: We demonstrated the possibility of highly effective correction of the F508del mutation in the CFTR gene in cells obtained from patients with CF using a designed system for editing this mutation based on the CRISPR/Cas9 system. Since hiBCs can be transfected and edited quite successfully, and can also be obtained in satisfactory quantities from hiPSCs, they are a promising platform for the development of gene therapy for cystic fibrosis.

BACKGROUND: The hypothesis that mitochondrial dysfunction may accompany development of chronic heart failure (CHF), type 2 diabetes mellitus (T2DM), and their comorbid forms is supported by real-world clinical observations. In patients with CHF with preserved ejection fraction (CHF-pEF) and reduced ejection fraction (CHF-rEF), as well as in patients with T2DM, mitochondrial stress test to assess mitochondrial respiration of peripheral blood mononuclear cells shows a significant decrease in oxygen consumption by mitochondria of peripheral blood mononuclear cells.

AIM: The aim of the study was to evaluate an informative value of the mitochondrial stress test in patients with CHF with T2DM.

MATERIALS AND METHODS: A total of 23 patients (mean age 69.8±10.1 years) with CHF-pEF and CHF-rEF were included. Patients were divided into groups according to the presence or absence of concomitant T2DM. A mitochondrial stress test was performed using the Seahorse XFe96 analyzer (Agilent Technologies, USA). Mitochondrial respiratory function was assessed in adherent mononuclear cells by simultaneous measurement of oxygen consumption and extracellular proton current flow.

RESULTS: In patients with T2DM, the basal respiratory capacity was reduced 1.5-fold and the reserve respiratory capacity was reduced 3.5-fold compared to the control group. The most inhibitory effect of T2DM on mitochondrial respiration was observed in the CHF-rEF group: 2.1 to 3.0 times lower compared to the control group. Concomitant T2DM was associated with a lower reserve respiration capacity which was also 2.4–4.5 times lower in patients with CHF alone and 18.0 times lower in patients with T2DM alone. In addition, T2DM patients showed a 1.28-fold suppression of non-mitochondrial respiration compared to the control group.

CONCLUSION: Significant mitochondrial dysfunction detected in comorbid patients is associated with the rapid clinical progression of CHF with T2DM and high incidence of decompensation. A decrease in basal and reserve respiratory capacity is a key factor in development of CHF in patients with T2DM.