Generation of iPSC line ICGi024-A from human skin fibroblasts of a patient with ring chromosome 18.

Ring chromosome 18 is a rare chromosomal disorders that usually originate de novo and correlate with clinical manifestation: developmental delay as well as microcephaly, brain and ocular malformations, hypotonia and skeletal abnormalities. We generate iPSC clonal cell line ICGi024-A with pluripotency properties which were demonstrated in vitro by three germ layer differentiation capacity. ICGi024-A can be used for disease modeling and fundamental investigation of ring chromosome instability.

Establishment of an induced pluripotent stem cell line (ICGi026-A) from peripheral blood mononuclear cells of a patient with fragile X syndrome.

Expansion over 200 CGG repeats in FMR1 gene causes inherited intellectual disability or autism spectrum disorder named as fragile X syndrome. Despite the known cause fragile X syndrome pathogenesis has not been specified yet. The ICGi026-A iPSCs line was obtained by the reprogramming of the peripheral blood mononuclear cells from a 9-year-old boy with fragile X syndrome. The ICGi026-A iPSCs expressed pluripotency markers, had a normal male karyotype (46, XY) and had the capacity to in vivo differentiate into the cells of three germ layers.

Establishment of an induced pluripotent stem cell line (ICGi025-A) from fibroblasts of a patient with 46,XY,r(8)/45,XY,-8 mosaicism.

Ring chromosomes are structural aberrations commonly associated with disease phenotype. We consider necessary to create the iPSCs with a ring chromosome 8, which can be used for disease modeling and related research. The ICGi025-A iPSCs line was obtained by the reprogramming of the skin fibroblasts from a 1-year-old boy with 46,XY,r(8)/45,XY,-8 mosaicism, developmental delay, microcephaly, dysmorphic features, diffuse muscle hypotonia, moderate proximal muscle weakness, feeding problems, and motor alalia. The iPSCs had expression of the pluripotency-associated markers. In vitro differentiated cells expressed the markers of the cells of three germ layers. That data allowed us to conclude that ICGi025-A cells were pluripotent.

Combined Effect of Bone Morphogenetic Protein-2 and Erythropoietin on Regeneration of Cranial Bone Defects in Mice.

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 µg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.

Recombinant Human Erythropoietin Proteins Synthesized in Escherichia coli Cells: Effects of Additional Domains on the in vitro and in vivo Activities.

The aim of this work was to compare biological activities of three variants of bacterially expressed human recombinant erythropoietin (EPO) with additional protein domains: 6His-s-tag-EPO protein carrying the s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) at the N-terminus and HBD-EPO and EPO-HBD proteins containing heparin-binding protein domains (HBD) of the bone morphogenetic protein 2 from Danio rerio at the N- and C-termini, respectively. The commercial preparation Epostim (LLC Pharmapark, Russia) produced by synthesis in Chinese hamster ovary cells was used for comparison. The EPO variant with the C-terminal HBD domain connected by a rigid linker (EPO-HBD) possesses the best properties as compared to HBD-EPO with the reverse domain arrangement. It was ~13 times more active in vitro (i.e., promoted proliferation of human erythroleukemia TF-1 cells) and demonstrated a higher rate of association with the erythropoietin receptor. EPO-HBD also exhibited the greatest binding to the demineralized bone matrix (DBM) and more prolonged release from the DBM among the four proteins studied. Subcutaneous administration of EPO-HBD immobilized on DBM resulted in significantly more pronounced vascularization of surrounding tissues in comparison with the other proteins and DBM alone. Therefore, EPO-HBD displayed better performance with regard to all the investigated parameters than other examined EPO variants, and it seems promising to study the possibility of its medical use.

Synthesis in Escherichia coli and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain.

Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.

Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domain Synthesized in E. coli: In Vivo Osteoinductivity in Experimental Models on Small and Large Laboratory Animals.

Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits.