Genes & Cells

Regenerative medicine uses cells as therapeutic agents to heal tissues and organs. It is a rapidly evolving area of research worldwide. Cell-based therapy has emerged as a pivotal treatment approach for articular cartilage defects, recognizing the limited regenerative potential of cartilage inherent to its structural biology. Given the inherent challenges associated with the standardization of cell-based drugs compared to conventional pharmaceuticals, the evaluation of their safety and efficacy in preclinical or clinical trials incurs particular considerations.

In the majority of cases, autologous chondrocytes and mesenchymal stem/stromal cells derived from various tissues become key components of cell-based therapies currently available for cartilage defects. The cell-based therapies that have been approved for clinical use vary in manufacturing methods, types of cells, and use of matrices as a cell carriers in the finished product. Furthermore, clinicians routinely use a range of surgical techniques to perform a biopsy procedure for the preparation and subsequent implantation of finished cell-based products. Each cell-based treatment option available for patients with cartilage diseases offers a particular indication, benefits, and limitations, underscoring the relevance of comparative analysis of the therapies currently used in clinical practice. This will facilitate clinicians in selecting the most suitable therapy, while researchers may potentially expand the range of diagnoses for such therapies or enhance their efficacy.

This review will focus on certain cell-based therapies that have currently arrived at the stages of clinical investigation and have been approved for the treatment of cartilage defects.

BACKGROUND: Somatic cloning of sheep is of great interest for genetic resource conservation, biomedicine, and biopharmaceuticals. However, its efficiency remains extremely low. One way to enhance the efficiency of this technology is to optimize its individual steps, particularly the procedure of oocyte enucleation and the transfer of somatic cell–derived cytoplasts into the perivitelline space — nuclear transfer. The chemical agent cytochalasin B enhances the oocyte’s resistance to external deformation and facilitates micromanipulation procedures. However, its application in cloning technology remains a subject of debate.

AIM: To evaluate the efficiency of somatic cloning in sheep (Ovis aries) using cytochalasin B during the preparation of matured oocytes prior to nuclear transfer, depending on the duration of this procedure.

MATERIALS AND METHODS: Nuclear transfer was performed using in vitro–matured oocytes containing a first polar body (PB1) in their perivitelline space. In the experimental group, oocytes with PB1 were incubated for 20 minutes in a medium containing 7.5 µg/mL cytochalasin B before nuclear transfer. The control group was maintained in an identical medium without cytochalasin B. Nuclear transfer was conducted using an inverted microscope equipped with a Narishige micromanipulation system (Narishige Scientific Inst. Lab., Japan). Oocyte chromosomes were removed blindly by aspirating the PB1 and the adjacent cytoplasm. Somatic cells were injected directly into the perivitelline space of the enucleated oocyte. Electrofusion was used to combine the oocyte–somatic cell complexes. The cytoplasmic hybrids formed as a result of electrical stimulation were activated using ionomycin, subjected to simultaneous treatment with 6-(dimethylamino)purine and cycloheximide, and then cultured for two days for embryonic development.

RESULTS: The nuclear transfer efficiency (the number of oocyte–somatic cell complexes relative to the number of oocytes with PB1) and fusion rate (the number of cytoplasmic hybrids formed relative to the number of oocyte–somatic cell complexes) did not differ between the control and experimental groups, remaining at 96%–97% and 33%–35%, respectively. In the control group, the proportion of cytoplasmic hybrids undergoing cleavage after two days of culture was 35.7±7.7%. Pre-incubation of oocytes in cytochalasin B increased this parameter to 59.7±6.9% (p <0.0029). However, the effect of cytochalasin B treatment on cloning efficiency depended on the duration of nuclear transfer: when the procedure exceeded 40 minutes, a significant decrease in the fusion rate (p=0.002) and a reduction in the proportion of cloned embryos (relative to the number of oocyte–somatic cell complexes) (p=0.041) were observed.

CONCLUSION: Short-term culture of matured oocytes in the presence of cytochalasin B before nuclear transfer increases the yield of cloned embryos, while prolonged nuclear transfer procedures negatively affect the efficiency of somatic cloning in sheep.

BACKGROUND: Activated stromal cells, responsible for tissue repair and restoration of tissue integrity, play an important role in tissue response to damage. The key marker of activated stromal cells is the fibroblast activation protein α (FAPα), which is apparently involved in the regulation of stromal cell functions, especially in the development of fibrosis. However, It is poorly known about tissue differences in expression and possible splice variants of this protein in stromal cells.

AIM: The aim is to establish the tissue specificity of FAPα expression, including the synthesis of alternative splicing products, in human stromal cells isolated from various sources.

METHODS: The expression of individual sites encoding the functional domains of FAPα in human stromal cells isolated from five tissue sources differing in embryonic origin and reparative reactions to damage (skin, subcutaneous adipose tissue, orbital adipose tissue, lungs, endometrium) was analyzed by real-time polymerase chain reaction. The key profibrotic factor TGFβ was used to activate stromal cells.

RESULTS: Low background expression of all RNA sequences encoding functional domains of FAPα responsible for protein configuration or for its enzymatic functions is noted in stromal cells from skin, lungs and adipose tissue. Their expression is significantly increased (2–2.5 times) upon induction of stromal cell activation by TGFβ. In endometrial mesenchymal stromal cell, the expression of these sites is extremely low and comparable to the level of expression in epithelial cells, which indicates the presence of tissue-specific expression of the FAPα gene in stromal cells. In all stromal cell lines, an increase in the relative expression level of the RNA sequence encoding the transmembrane domain of FAPα is observed.

CONCLUSION: Thus, the obtained results suggest that the analyzed cell lines either lack alternative FAPα splicing, or manifest themselves with a very low probability when activated in the form of a relative increase in the expression of the gene region encoding the transmembrane domain of FAPα. Most probably, the studied stromal cells synthesize mRNA carrying all the original exons and encoding predominantly the complete FAPα protein, that includes all functional domains.