It has been established previously that up to 40% of mouse CD34+ hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.

We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.

The levels of 17 cytokines in the follicular fluid were measured by multiplex proteome analysis at the peak of stimulated ovulation during the in vitro fertilization cycle. In patients with ineffective folliculogenesis, the concentrations of IL-2, IL-4, IL-7, and granulocytic CSF in the follicular fluid were significantly lower than in women with greater mean number of collected oocytes. It was shown that multicomponent cytokine deficit was associated with lower production of high quality oocytes and lower efficiency of fertilization of the resultant oocytes in vitro. The absence of pregnancy after in vitro fertilization cycle is associated with lower levels of IL-2, IL-4, IL-7, granulocytic CSF, and macrophagic inflammatory protein 1b in combination with elevated contents of IL-8 and IL-13. The results attest to an important role of cytokines in the regulation of oogenesis and in preparation of the endometrium to implantation of the embryo.

Comparative analysis of mesenchymal stromal cells isolated from human BM, adipose tissue, and placenta was carried out. The cells were compared by the levels of constitutive, spontaneous, and LPS-induced production of Th1/proinflammatory (IFN-γ, IL-2, IL-1β, TNF-α, IL-12, IL-17) and Th2/anti-inflammatory cytokines (IL-4, IL-5, IL-6, IL-10, IL-13), chemokines (IL-8, MCP-1, MIP-1β), growth factors (IL-7, granulocytic CSF, granulocytic macrophageal CSF, erythropoietin, VEGF, EGF, IGF-1, main FGF), matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Mesenchymal stromal cells originating from different tissues were characterized by functional potential for hemopoiesis support (through production of granulocytic CSF, granulocytic macrophage CSF, erythropoietin), immunomodulation (through production of IFN-γ, IL-2, IL-6, IL-1β, TNF-α and chemokines IL-8, MCP-1, MIP-1β), and stimulation of reparative processes (through production of VEGF, FGF, IGF-1, IL-6 tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-9). By the type and levels of spontaneous (basal) production of cytokines, the adipose tissue mesenchymal stromal cells more distinctly demonstrated the proinflammatory (IL-1β TNF-α), immunoregulatory (IFN-γ, IL-2, IL-4, IL-6, IL-8, MCP-1, MIP-1β), and hemopoiesis-stimulating (granulocytic CSF, granulocytic macrophage CSF) phenotype and at the same time were characterized by lower sensitivity to lipopolysaccharide stimulation than BM and placental mesenchymal cells.

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Stem cell-based therapy is considered to be a new approach for the treatment of cerebral palsy (CP). Given the potent anti-inflammatory activity and high regenerative potential of M2 macrophages, these cells may be an alternative source for cell transplantation. To evaluate the safety and efficacy of autologous M2 macrophages, we conducted a pilot clinical trial in 21 children with severe CP. The primary outcome measure was safety, which included assessment of mortality of any cause, immediate adverse reactions, and serious adverse effects and comorbidities during 5-year follow-up. The secondary outcome measure was functional improvement in Gross Motor Function Measure (66-item GMFM) test, Peabody Developmental Motor Scale-Fine Motor (PDMS-FM) test, Ashworth scale, MRC scale, and an easy-to-understand questionnaire for evaluation of cognitive functions in our modification. Intradural injection of M2 cells (in mean dose of 0.8 × 10(6)/kg) into the lumbar spinal area did not induce any serious adverse events. No cases of mortality, psychomotor worsening, exacerbation of seizures, and long-term comorbidities, including tumors, were observed during a 5-year follow-up. After 3 months, GMFM score increased from 13.7 ± 7.8 to 58.6 ± 14.6, PDMS-FM score improved from 0.76 ± 0.42 to 5.05 ± 0.97, and the Ashworth score decreased from 3.8 ± 0.21 to 3.3 ± 0.24. Along with gross and fine motor function enhancement, an improvement of cognitive activity (from 1.62 ± 0.41 to 4.05 ± 0.64, according to questionnaire assessment) and reduction of seizure syndrome were registered as well. The neurological improvements did not diminish during the 5-year follow-up period. The data obtained suggest that cell therapy based on M2 macrophages is safe, does not induce early adverse effects and long-term comorbidities, and is accompanied with a significant improvement of motor and cognitive activities in severe CP patients. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

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