RU2732599C1 - Nootropic composition based on polypeptide complexes isolated from glial progenitor cells under conditions of heat shock, and a method for production thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to production of a nootropic composition based on polypeptide complexes recovered from glial progenitor cells in conditions of heat shock. Method involves sampling a skin biopsy material, obtaining a fibroblast culture, cryopreservation of a fibroblast culture, defrosting the fibroblast culture, adapting the fibroblast culture to xeno free cultivation conditions, reproducing the fibroblasts for producing induced pluripotent stem cells (iPSC), iPSC cultivation, iPSC cryopreservation, iPSC defrosting, iPSC differentiation into glial progenitor cells, cultivation of glial progenitor cells under heat shock conditions, collecting the conditioned medium and obtaining a protein-peptide complex. To simulate heat shock, glial precursors are cultured for hour at 40 °C in growth medium containing DMEM/F12, 2 % additive B27, 20 ng/ml FGF-2, 1 mcM purmorfamin, after which the medium is replaced with DMEM/F12 and cultivated for 6 hours, after which the conditioned medium is collected and centrifuged at 300 rpm for 5 minutes, supernatant is collected and concentrated 24 times with 3 kDa Amicon Ultra membranes, then the obtained concentrate is subjected to sterilizing filtration to obtain a nootropic composition. Composition contains proteins and polypeptides with molecular weight from 3 to 250 kDa, of which 65 % have a molecular weight in range of 10 to 60 kDa, content of heat shock proteins of not less than 40 % of total protein weight, brain-derived neurotrophic factor (BDNF) of not less than 140 pg/ml and glial cell-derived neurotrophic factor (GDNF) of not less than 60 pg/ml, vascular endothelium growth factor (VEGF) of not less than 80 pg/ml, total protein weight of not less than 1 mg/ml of preparation, wherein the cell culture of glial progenitor cells from which the composition is obtained demonstrates the expression of astroglial markers S100b and GFAP and consists of 98±2 % S100b⁺ cells.

EFFECT: invention widens the range of equipment.

2 cl, 2 dwg, 4 ex

Description

In the treatment of diseases of the central nervous system, great hopes are placed on technologies based on the use of induced pluripotent stem cells (iPSCs), which can be obtained from a mature somatic cell by transduction with certain genes [Nekrasov E.D. et al., 2011]. The use of these technologies makes it possible to obtain autogenous neuronal and astroglial precursors for use in the complex treatment of neurodegenerative diseases. For the development of treatment methods using iPSCs and the introduction of products based on them into clinical practice, experimental and preclinical studies are needed to elucidate the molecular mechanisms of action, determine the cell populations with the greatest therapeutic effect, study dose-dependent effects, frequency of administration, etc. [Kim S.U. et all, 2009]. Modern protocols for iPSC differentiation make it possible to obtain both neurons and glial cells [Chambers S.M. et all 2009; Madhu V. et all, 2016; Wechsler-Reya R. J. et all, 1999; Hughes S.M. et al., 1988; Song M.R., 2004]. Until recently, the main focus in the treatment of neurodegenerative diseases was on neuroprotection, since it was believed that regeneration does not occur in the central nervous system. However, in recent years, studies have begun to appear that indicate reparative processes in the central nervous system in the postnatal period. It turned out that in the brain there are neural stem cells (NSC), which are concentrated in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus [Eriksson, P.S. et all 1998]. Brain damage has a stimulating effect on the proliferative activity of NSCs, their migration to the damaged area and subsequent differentiation into neuronal and glial cells, the replacement capacity of which is only 0.2% of the number of dead cells [Mattson M.R. et all, 2000; Kernie S.G. et all, 2010]. The next stage of the reparative process is the reorganization of the neural network due to the plasticity of neurons. This process is characterized by a change in the number and length of neuronal processes and the establishment of new synaptic connections [Castren E. Et all, 2017]. An integral part of the reparative processes is also the restoration of the BBB and neovascularization [Xiong, Y. et all, 2010; Prakash R. et all, 2015]. These facts about the ability of the central nervous system to repair allow us to conclude that regeneration is an important mechanism of neurological recovery after injury and is a new target of therapeutic interventions. Cell therapy, as a new direction in the treatment of neurodegenerative diseases, served as the basis for the active development of new therapeutic strategies for treating stroke based on the use of stem cells (SC). However, cell technologies have been criticized for a long time due to the lack of a single specific target and understanding of the mechanisms of action of a cell transplant. To date, there is a lot of data on the use of postnatal bone marrow cells, such as hematopoietic stem cells, mesenchymal stem cells, endothelial progenitor cells, as well as fetal stem cells, which have a therapeutic effect in neurodegenerative diseases [Quittet M.S. et al., 2015; Giraldi-Guimaraes, et all, 2009; Napoli E. et all, 2016]. In recent years, the ability of stem cells to secrete biologically active substances has been demonstrated that have a positive effect on the course of many neurodegenerative diseases. Conditioned media (CC) are a source of a large number of paracrine factors, including neurotrophins (BDNF, GDNF, NGF, NT3-5, CNTF, etc.), cytokines [Prospect of Stem Cell Conditioned Medium in Regenerative Medicine. Jeanne Adiwinata Pawitan. BioMed Research International 2014, Article ID 965849], exosomes [Microvesicles from brain-extracttreated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke. Ji Yong Lee, Eiru Kim, Seong-Mi Choi, Dong-Wook Kim, Kwang Pyo Kim, Insuk Lee & Han-Soo Kim. Scientific Reports. Published: 09 September 2016] and are capable of providing a complex effect for various pathologies. There are results of numerous studies indicating the importance of the role of neurotrophins in the development of Alzheimer's disease, and their ability to provide neuroprotective, antiapoptotic, neurotrophic effects. Cytokines in the CS are capable of anti-inflammatory effects. Exosomes, also contained in CS, can influence the elimination of beta-amyloid from brain tissue [Exosomes as Carriers of Alzheimer's Amyloid-β. Kohei Yuyama and Yasuyuki Igarashi Front. Neurosci., 25 April 2017]. It was shown that CS of neural stem cells (NSC) inhibited apoptosis of SH-SY5Y cells in culture, and also increased the percentage of differentiated cells when combined with retinoic acid [Human neural stem cell transplantation improves cognition in a murine model of Alzheimer's disease. Lisa M. McGinleyl, Osama N. Kashlan2, Elizabeth S. Brunol, Kevin S. Chen2, John M. Hayesl, Samy R. Kashlan1, Julia Raykin3, Karl Johe4, Geoffrey G. Murphy5 & Eva L. Feldmanl. Scientific Reports, Article number: 14776 (2018)].

There are data confirming the effectiveness of CS in in vivo models for neurological diseases, such as ischemic stroke - when CS NSC was administered to rats with simulated ischemic stroke, the indicators for assessing the neurological status were higher than in the control group, the volume of infarction decreased compared to the control, so the change in the level of apoptotic activity was investigated by the TUNEL method, which also showed a decrease in cell death [Neural Stem Cell-Conditioned Medium Ameliorated Cerebral Ischemia-Reperfusion Injury in Rats HongNa Yang, Cuilan Wang, Hui Chen, Lan Li, Shuang Ma, Hao Wang, YaRu Fu, and Tingyu Qu. Stem Cells International. Published April 2018]. In the model of spinal trauma, the effect of CS NSC on the viability of neurons and regeneration of axons of the corticospinal tract was studied. The use of CS led to extensive growth of axons in the cervical spine and more than tripled the formation of synaptic contacts between collaterals of the corticospinal tract and propriospinal interneurons. CS also reduced caspase 3 expression and decreased neuronal death 6 weeks after injury [Neural Stem Cell-Conditioned Medium Protects Neurons and Promotes Propriospinal Neurons Relay Neural Circuit Reconnection After Spinal Cord Injury. Peng Liang, Jiaren Liu, Jinsheng Xiong, Qing Liu, Jiaxin Zhao, Hongsheng Liang, Liwei Zhao, and Haitao Tang. Cell Transplantation, Vol. 23, Supplement 1, pp. S45-S56, 2014]. The conditioned medium of adipose tissue mesenchymal stem cells in the tail suspension model restored the reduced immobility time in 5xFAD transgenic mice to normal levels, thus exerting an antidepressant effect by inhibiting the Akt signaling pathway and activating GSK-3β [Adipose-derived stem cell-conditioned medium ameliorates antidepression-related behaviors in the mouse model of Alzheimer's disease. Hiromitsu Yamazaki, Yu Jin, Ayako Tsuchiya, Takeshi Kanno, Tomoyuki Nishizaki. Neuroscience Letters. Available online 17 October 2015]. CS of mesenchymal stem cells of the dental pulp was tested on a model of exogenous amyloidosis - intranasal administration of CS of this type of cells led to a significant improvement in cognitive functions. Western blotting and real-time PCR were used to assess markers responsible for neuroprotection, neuritogenesis and anti-inflammatory effect on microglia [Conditioned medium from the stem cells of human dental pulp improves cognitive function in a mouse model of Alzheimer's disease. Tsuneyuki Mitaa, Yoko Furukawa-Hibib, Hideyuki Takeuchic, Hisashi Hattoria, Kiyofumi Yamadab, Hideharu Hibia, Minoru Uedaa, Akihito Yamamotoa, Behavioural Brain Research. Available online 22 July 2015].

Also, clinical trials of the peptide preparation Cerebrolysin containing amino acids and polypeptides with a molecular weight of up to 10 kDa indicate its effectiveness in vascular dementia [Cerebrolysin for vascular dementia. Chen N, Yang M, Guo J, Zhou M, Zhu C, He L. Cochrane Database Syst Rev. 2013 Jan 31]. Moreover, recently there have been data on the neuroprotective properties of heat shock proteins and their possible secretion from cells into the environment. Presumably, the secretion of heat shock proteins by stem and progenitor cells when exposed to elevated temperatures should enhance the neuroprotective effect.

At present, iPSCs are considered as the most promising source of neural precursors for regenerative medicine, which makes it possible to obtain cell populations of various types at different, pre-planned stages of embryonic development. And at the same time, the problems of shortage of donor material and ethical issues arising from the use of embryonic material and cultures of embryonic stem cells are completely absent. The production of iPSCs has already become a routine task, although experimental studies have been carried out only for the last 10 years. As a result of experiments on retroviral transduction by genes of various transcription factors of mouse fibroblasts, 4 genes - Oct-4, Sox2, c-Myc, and Klf4 - were identified, which transferred them to a pluripotent state. The cells obtained in this way were called induced pluripotent stem cells (iPSCs) [Takahashi K., Yamanaka S. 2006]. Then iPSCs were successfully obtained from human fibroblasts using the same set of transcription factors [Takahashi K. et al., 2007]. Another scientific group obtained human iPSCs using a different set of transcription factors: Oct4, Sox2, Nanog and Lin28 [Yu J. et al., 2007]. Further studies have demonstrated the possibility of obtaining iPSCs both from various mammals: rats [Li W. et al., 2009], rhesus monkeys [Liu N. et al., 2008], pigs [Kwon D.-J. et al., 2013]; and from various types of human somatic cells: keratinocytes [Aasen T. et al., 2008], neuronal cells [Eminli S. et al., 2008.], liver and stomach cells [Aoi, 2008], terminally differentiated lymphocytes, as well as from the endothelium [Lagarkova MA et al., 2010]. These studies demonstrated the versatility of the process of reprogramming somatic cells to a pluripotent state by introducing transcription factor genes into cells. A variety of approaches have been developed to deliver reprogramming factors to somatic cells. Were also demonstrated other methods of reprogramming somatic cells to a pluripotent state without integrating exogenous DNA into the genome of the host cell: using piggyBac transposon [Woltjen K. et al., 2009], synthetic RNA, adenoviral vectors [Zhou W., Freed CR, 2009], Sendai virus [Fusaki N. et al., 2009], episomal vectors [Yu J. et al., 2009], proteins [Kim D. et al., 2009]. Differentiation tests are considered key in proving cell pluripotency. For human iPSCs, in vitro differentiation, the formation of teratomas when administered to immunodeficient mice, and the ability to form embryoid bodies are used as the main tests. Pluripotent stem cells (PSCs) naturally differentiate into all types of adult cells, but the most important experimental task is to differentiate pluripotent cells in vitro in the desired direction using special cultivation conditions, biologically active chemical compounds, and protein factors. An effective method of iPSC differentiation in the neural direction is based on blocking BMP and TGF-β signaling pathways by molecules such as Noggin, dorsomorphin, SB431542 and LDN193189 at the early stages of differentiation [Fingar D. C, et al., 2004]. This approach, during long-term cultivation, made it possible to obtain up to 90% of cells that expressed the neuroectoderm markers PAX6 and PSA-NCAM (nerve cell adhesion molecule). For further differentiation into neuronal progenitors, activation of the Shh signaling pathway is required, as well as the addition of fibroblast growth factor FGF2 to the culture medium. The precursors thus obtained express the immature neuron marker β-tubulin III. The routine method of obtaining iPSCs, as well as the existence of known methods for differentiating iPSCs in the neural direction, make the use of neuronal precursors the most promising candidates for IS therapy. The possibility of effective expansion of iPSCs will solve the problem of the lack of donor material and remove a number of ethical issues. Today, a large number of research groups are developing in the field of neural differentiation of iPSCs. The main leaders in this area are the Institute of Genetic Diseases and Genomic Science, The Jikei University School of Medicine, Tokyo, Japan; Center for iPS Cell Research and Application, Kyoto University, Japan; Center for Cell Biology, China (Department of Cell Biology, Xuanwu Hospital of Capital Medical University, Beijing, China). These laboratories conduct cutting-edge research on iPSC differentiation, rationalize and create iPSC differentiation protocols. In addition, experimental developments are underway to introduce neurally differentiated iPSCs into target organs. In this regard, it is worth noting the Laboratory of the Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore, which carries out innovative developments in the field of iPSC differentiation, and methods of targeted delivery of differentiated cells to target organs. , using 3D microfiber capsules that increase delivery efficiency. European laboratories and laboratories in the USA and Canada occupy a leading position in the total number of research works in the field of neural differentiation of iPSCs. When studying the literature, modern developments of the laboratory of the Genetics Center of the University of St. Wisconsin in collaboration with the Institute for Biomedical Research, Cambridge (Waisman Center, Genome Center, Department of Anatomy, and Department of Neurology, School of Medicine and Public Health, University of Wisconsin, Madison. Whitehead Institute for Biomedical Research, Cambridge). In these works, one can note the advanced research on the search for new regulatory mechanisms in the field of neural differentiation of iPSCs.

The main reason limiting the effectiveness and safety of all existing neuropeptide drugs derived from the mammalian brain is their xenogenic origin. When isolating neuropeptides from the brain tissues of animals to reduce their immunogenicity (antigenic properties), they resort to acidic or enzymatic hydrolysis of proteins and high molecular weight polypeptides, thereby reducing the amount of neurotrophic growth factors of protein and polypeptide nature [Khabrieva. U., 2005]. The peptide preparation obtained in this way contains denatured products of acidic hydrolysis of proteins and polypeptides with a disturbed quaternary and tertiary structure, which reduces its specificity and biological activity, increases its immunogenicity, causing allergic reactions when applied.

To solve this problem, we proposed to obtain a protein-peptide composition with pronounced nootropic properties from the secretome of glial progenitor cells under hypoxic conditions, obtained by the method of directed differentiation of induced human pluripotent stem cells. This composition is characterized by the fact that it contains proteins and polypeptides with a molecular weight from 3 kDa to 250 kDa, of which 65% have a molecular weight in the range from 10 to 60 kDa, the content of heat shock proteins is not less than 40% of the total mass of proteins , brain neurotrophic factor (BDNF) not less than 140 pg / ml and glial neurotrophic factor (GDNF) not less than 60 pg / ml, vascular endothelial growth factor (VEGF) not less than 80 pg / ml, total protein mass not less than 1 mg / ml preparation, while the cell culture of glial progenitor cells, from which the composition is obtained, demonstrates the expression of astroglial markers S100b, GFAP and consists of 98 ± 2% S100b ⁺ - cells.

The most important area of modern medicine and biology is the isolation of biologically active substances from cell cultures, primarily molecules that trigger a cascade of regeneration processes and paracrine induction of reparative processes. The use of a characterized and standardized complex of biologically active substances can have the same efficiency, but much greater safety compared to living cell cultures. The possibility of effective expansion of iPSCs and their differentiation in different directions, including neuronal, solves the problem of lack of donor material and removes a number of ethical issues.

The invention is carried out as follows.

Example 1. Obtaining a protein-peptide complex.

A skin biopsy sample (0.5 to 1 cm ² ) is taken behind the auricle or on the inside of the forearm. After cutting with a microsurgical blade, the biopsy specimen is placed in a sterile vial containing a transport medium (F12 with amikacin, 0.5 g / L). The vial with the contents is marked with the following data: name of the donor, date and time of collection. Transportation is carried out at a temperature of 2-6 ° C.

The resulting material is transferred under sterile conditions into Petri dishes and washed three times with Hanks solution with cefazolin (1 g / l), then disaggregated by incubation in Versene solution with the addition of 0.25% trypsin. Within 1-1.5 hours at 37 ° C. After incubation, the suspension is vigorously mixed and centrifuged for 10 minutes at 1400 rpm. The precipitate is diluted with culture medium (DMEM / F12 1: 1 with the addition of 10% serum, 500 mg / l amikacin), mixed and transferred to culture dishes. The culture flasks are placed in a CO ₂ incubator (37 ° C, 5% CO ₂ ). After 2-3 days, the culture medium is replaced with a fresh one. Growing and reseeding is carried out with Versene solution with the addition of 0.25% trypsin and transferred to new Petri dishes. The seeding ratio is selected depending on the individual proliferative characteristics of fibroblasts. No more than four passages are cultivated. Before programming, it is necessary to control the stability of the karyotype and the ability of fibroblasts to synthesize type I collagen (using immunofluorescent staining).

If long-term storage is required, the resulting fibroblasts are cryopreserved. In a box in an ice bath (at 4 ° C), a cryoprotective solution is prepared, consisting of 10% DMSO (dimethyl sulfoxide) and 90% autogenous serum. The cell culture is disaggregated with a Versene solution with the addition of 0.25% trypsin, the number of cells is counted using a counter and the cells are pelleted by centrifugation (1400 rpm, 10 min). A freshly prepared cryoprotective solution is added to the sediment at the rate of 1 ml per 5 million cells, mixed and the resulting suspension is placed in cryovials. Mark, indicating the name of the donor, the passage number, the number of cells, the date of cryofreezing, the data of the responsible executor.

Samples are frozen to -80 ° C using a software freezer, and then placed in Dewar flasks with liquid nitrogen and stored at -196 ° C.

For defrosting, cryotubes are removed from the Dewar flask and thawed at 37 ° C in a water bath. The cell suspension from the cryotube is transferred into a centrifuge tube with a culture medium filled to ten times its volume and centrifuged at 1400 rpm for 10 minutes. The cell pellet is transferred to a culture dish with complete growth medium.

Before the reprogramming procedure, the culture of fibroblasts is transferred to a medium that does not contain components of animal origin. For this, according to the protocol, fibroblasts 1-2 passages are washed with phosphate-buffered saline and treated with Trypsin / Versene 1: 1 solution for 3-5 minutes to detach the cells from the substrate. The cell suspension is centrifuged for 5 minutes at 1100 rpm, after which the supernatant is taken, and DMEM medium containing 10% fetal bovine serum, 2 mM L-glutamine, penicillin-streptomycin 100 mg / l is added to the sediment and seated in an amount of 1500-2000 cells / cm ² on Petri dishes 60 mm in diameter, coated with the Coating Matrix Kit collagen (Corning, USA). After 24 hours, the adherent cells are washed twice with phosphate-buffered saline and a medium consisting of DMEM / F12, HEPES, sodium bicarbonate 4.8 g / ml, 10% serum substitute Knockout SR, human fibroblast growth factor bFGF 10 μg / ml, epidermal human growth factor EGF 1 mg / ml, 2 μM hydrocortisone. To adapt cells to a new environment, cultivation is carried out for 1-2 passages.

For reprogramming fibroblasts, a commercial kit for reprogramming human cells "CTS CytoTune-iPS 2.1 Sendai Reprogramming Kit" (bivitrogen, USA) based on the Sendai virus is used. The day before reprogramming, fibroblasts are seeded in an amount of 200 thousand per well of a 6-well plate, previously covered with collagen, and cultured in 2.5 ml of medium without exogenous components. The next day, vectors are added carrying the genes Oct4 and Sox2 (KOS), L-Myc (hL-Myc), Klf-4 (hKlf4) in the number of viral particles per cell (MOI) - 5, 5 and 3, respectively. A day later, the medium for fibroblasts is replaced without exogenous components, then the medium is changed daily for 6 days. On the 7th day after reprogramming, the cells are washed twice with PBS, detached with TrypLE Select reagent and centrifuged at 1100 rpm for 5 minutes. The resulting supernatant was removed, and the cell pellet was transplanted in the amount of 100 thousand per Petri dish 60 mm in diameter, previously coated with vitronectin at a concentration of 10 μg / ml. 24 hours after seeding, the medium is replaced with Essential 8 Medium for the cultivation of human pluripotent cells. The medium is changed every day.

The selection of the formed colonies is carried out according to the results of in vivo immunocytochemical staining with antibodies to the surface marker of pluripotent cells - proteoglycan TRA-1-60 using a commercial set "TRA-1-60 Alexa Fluor 488 Conjugate Kit" (ThermoFisher Scientific, USA). Colonies of iPSCs, in which high expression of the proteoglycan TRA-1-60 was visualized, were selected mechanically with a tip, pretreated with a Versene solution, then planted on 6-well plates coated with vitronectin and cultured in Essential 8 Medium. Further subcultures are carried out only with the Versene solution and with the addition of the ROCK inhibitor Y27632 to the medium at a concentration of 5 μM during the day.

During the cultivation of iPSCs, visual control over the morphological characteristics of the cultures is carried out. IPSCs grow in dense colonies with smooth edges. The cells are small, with large nucleoli. Culturing is carried out in 35 mm Petri dishes using Essential 8 ™ culture medium (E8).

The cells are planted after reaching a density of 50-70% of the monolayer. The culture medium is removed from the plates and washed with 2 ml DPBS. For disintegration, add 1 ml of Versene solution and incubate for 5-8 min at room temperature. Then remove the Versene solution, add 2 ml of medium, shake and transfer the cell suspension to dishes previously coated with vitronectin, and add the medium to 2 ml. The medium is replaced with a fresh one every day.

Standardization of iPSCs is carried out by the expression of genes specific for embryonic stem cells OCT4, SOX2, NANOG, SSEA4, TRA-1-81, TRA-1-60 by immunocychemistry and PCR with reverse transcription

IPSCs are cryopreserved. A cryoprotective solution is prepared on ice at the rate of 1 dish: 900 μl of E8 medium, 100 μl of DMSO and 1 μl of Rho-kinase inhibitor (5 mM stock). Remove medium from each plate and wash with 2 ml DPBS. Add 1 ml of Versene and incubate for 5-8 min at room temperature. Then remove the Versene solution and add 1 ml of cryoprotective solution. The cells are removed from the surface of the plate, mixed and transferred to a 1.8 ml cryovial (1 ml per tube). Samples are frozen to -80 ° C using a software freezer, and then placed in Dewar flasks with liquid nitrogen and stored at -196 ° C.

Cryotubes with cellular material are removed from the Dewar flask and thawed at 37 ° C in a water bath.

The cell suspension from a cryovial (1 ml) is transferred dropwise into a centrifuge tube, 9 ml of warm DMEM / F12 culture medium is added to tenfold volume and centrifuged at 800 rpm for 5 minutes.

The cells washed from the cryopreservative are transferred onto Petri dishes, previously coated with vitronectin, with the E8 culture medium containing the Rho kinase inhibitor. During the transfer, the integrity of the colonies is preserved. Cell adaptation after cryopreservation lasts from 1 to 3 weeks.

For differentiation in the astroglial direction, iPSCs are treated with Versene's solution, centrifuged at 800 rpm for 5 minutes. The supernatant was removed, and the cell pellet was transferred to new 60 mm Petri dishes coated with 20 μg / ml poly-1-ornithine (Sigma-Aldrich, USA) and 2.5 μg / ml laminin (Gibco, USA). When the culture reaches 80% confluency, Essential 8 Medium is changed to DMEM / F12 with 1% N2, 2 mM glutamine, penicillin-streptomycin 100 mg / l, 10 μM SB431542, 2 μM dorsomorphin. Wednesday is changed every other day. After 10-14 days, rosette-like structures appear in the culture. After, the resulting culture is subcultured on Petri dishes coated with poly-l-ornithine 20 μg / ml and laminin 2.5 μg / ml, at a density of 250-400 thousand cells / cm ² and cultured in DMEM / F12 medium, 1% N2 additive , 2 mM glutamine, 100 mg / L penicillin-streptomycin, 1% serum (Paneco, RF), 10 ng / ml FGF-2, 20 ng / ml EGF (ProSpec, UK) for three days. Then the medium is replaced with DMEM / F12, 1% serum, 1% N2 supplement, 2 mM glutamine, 100 mg / L penicillin-streptomycin (Paneco, RF), 10 ng / ml FGF-2, 20 ng / ml EGF (ProSpec, UK), 20ng / ml CNTF (PeproTech, USA). For the next three days, cells are cultured in DMEM / F12 medium, 1% serum, 1% N2 supplement, 2 mM glutamine, 100 mg / L penicillin-streptomycin (Paneco, RF), 20 ng / ml EGF (ProSpec, UK), 20ng / ml CNTF (PeproTech, USA).

Glial progenitor cells are standardized by expression of genes S100b, GFAP, and CD44 (a marker of fibrous astroglia).

To simulate heat shock, glial progenitors are cultured for one hour at 40 ° C., after which the medium is replaced with DMEM / F12 and cultured for 6 hours. Then the conditioned medium is collected and centrifuged at 300 rpm for 5 minutes. The supernatant was removed and concentrated 24-fold using 3 kDa Amicon Ultra membranes (Sigma-Aldrich, USA). The resulting concentrate is subjected to sterilizing filtration. The composition includes proteins and peptides with a molecular weight ranging from 3 to 250 kDa, the average molecular fraction is in the range from 10 to 60 kDa, not less than 65%.

Example 2. Characterization of glial progenitor cells.

To confirm the phenotype of the obtained cultures of glial progenitor cells, immunocytochemical staining for glial markers is performed. The cells are fixed with 4% formaldehyde solution (Merck KGaA, Germany) for 10 minutes at room temperature, then washed with phosphate-buffered saline, incubated in phosphate-buffered saline with 0.25% Triton X-100 (Sigma-Aldrich, USA) and 1% bovine serum albumin solution (AppliChem, Germany) for 30 minutes, and then treated with primary antibodies overnight at + 4 ° C. For glial stem cells, antibodies to proteins S100b, GFAP and CD44 (a marker of fibrous astroglia) are used. Then the cells are washed with phosphate-buffered saline and incubated with secondary antibodies labeled with fluorochromes Alexa Fluor 488 anti-rabbit (dilution 1: 600, Invitrogen, USA), Alexa Fluor 555 anti-mouse (dilution 1: 600, Invitrogen, USA) in the dark 60 minutes. The nuclei were stained with a solution of DAPI (4,6-diamino-2-phenylindole dihydrochloride) 1 μg / ml (Sigma-Aldrich, USA) in phosphate-buffered saline. An Axio Observer D1 inverted luminescence microscope with an AxioCam HRc camera (Carl Zeiss, Germany) was used for the analysis.

Quantification is carried out using flow cytometry. The culture is removed using 0.05% trypsin solution (Paneko, RF), centrifuged at 1600 rpm for 5 minutes, the supernatant is removed, and the precipitate is washed with Hanks solution. After sedimentation, cells are fixed for 10 minutes with 4% paraformaldehyde solution, then cells are washed with phosphate-saline solution. Permeabilization is carried out with 70% methanol on ice for 10 minutes, after which the cells are washed twice in phosphate-saline solution and pelleted by centrifugation at 1800 rpm for 5 minutes. Then the cells are stained with primary antibodies against S100b (dilution 1: 200, Abcam), incubation took place at + 4 ° C for 12 hours. Then it was washed with phosphate-buffered saline, centrifuged at 1800 rpm for 5 minutes and incubated with secondary antibodies labeled with Alexa Fluor 555 anti-mouse fluorochrome (dilution 1: 600, Invitrogen, USA) in the dark for 60 minutes. The resulting suspension was analyzed using a CyFlow ML flow cytometer, and the number of S100b ⁺ cells was counted using the FloMax software. According to flow cytometry data, the culture of neuronal progenitor cells should consist of at least 98 ± 2% S100b ⁺ cells.

To analyze the expression of glial marker genes, PCR was performed. mRNA was isolated using a commercial RNeasy Mini Kit according to the manufacturer's instructions. Next, cDNA synthesis was carried out using the RevertAid ™ First Strand cDNA Synthesis Kit according to the protocol attached to the instructions. For real-time PCR, a ready-made reaction mixture with an intercalating dye Sybr Green was used; amplification was performed in a BioRad iQ cycler thermal cycler. Reaction scheme: primary denaturation 95 ° С, 5 min; denaturation 95 ° С, 20 sec .; annealing of primers 55-63 ° С, 20 sec .; elongation 72 ° C, 20 seconds (40 cycles). The mRNA level of the analyzed genes was leveled with respect to the averaged data of amplification of two housekeeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ActB (β-actin). The relative amount of mRNA was calculated using the ΔC (T) method.

As a result of differentiation, glial progenitor cells showed expression of the marker genes S100b, GFAP, and CD44 (Figure 1) both at the level of transcription and translation. The analysis showed that up to 98 ± 2% of the resulting cells expressed the glial marker S100b (Figure 2).

Example 3. Characteristics of the nootropic composition

The composition was characterized by the amount in 1 ml of total protein of such factors as Brain-Derived Neurotrophic Factor (BDNF), glial neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), and the amount of heat shock proteins. For this, an enzyme-linked immunosorbent assay was used. 1 ml of the suspension was centrifuged, the supernatants obtained as a result of centrifugation were applied to the wells of the plate with pre-adsorbed primary antibodies to BDNF, GDNF, VEGF and heat shock proteins (Hsp60, Hsp70, and Hsp90), then the analysis was performed according to the manufacturer's instructions. The content of factors was expressed in pg / ml of the composition. In total, 10 samples of the composition were examined, the results were obtained on the content of BDNF not less than 140 pg / ml, GDNF not less than 60 pg / ml, VEGF not less than 80 pg / ml, Hsp60 not less than 20,000 pg / ml, Hsp70 not less than 15,000 pg / ml and Hsp90 not less than 6000 pg / ml. The total mass of protein is not less than 1 mg / ml of the drug. Total protein in 1 ml of suspension was determined by the Bradford method.

Example 4. Evaluation of the effectiveness of using the composition in an in vitro model of chemical hypoxia on the SHSY-5Y cell line

To study the neurotrophic and neuroprotective effects of HP, the SHSY-5Y neuroblastoma line, which is widely used as an in vitro model in neurobiology, was selected. [eleven]. SHSY-5Y cells (ATCC, USA) were cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS). One week before the experiment, the cells were subcultured into 12-well plates coated with a type I collagen solution in the amount of 90,000 cells per well and then incubated in Opti-MEM medium containing 10 mM retinoic acid and 0.5% PBS for two days. Then the medium was changed to DMEM / F12 with the addition of 50 ng / ml of the neurotrophic factor BDNF and 2% B27. The neurotrophic factor BDNF was removed from the medium 72 hours before the simulation of hypoxia.

We used a model of chemical hypoxia, which is adequate and interchangeable with the model based on the use of a gas incubation chamber with a reduced oxygen content. To simulate hypoxia, cobalt dichloride CoCl ₂ was tested at concentrations from 150 to 300 μM. SHSY-5Y neuroblastoma cells were cultured in 12-well plates with CoCl ₂ for 4 hours, after which they were washed with Hanks solution and DMEM / F12 medium with 2% B27 was added. To study the paracrine effect, after the simulation of hypoxia, co-cultivation with HP was performed using a Transwell insert, which were incubated in DMEM / F12 medium supplemented with B27.

To determine the viability of the SHSY-5Y cell line, after 24 hours of co-cultivation, MTT (3- [4,5dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide, PanEco, RF) was added at a concentration of 0.5 mg / ml and incubated in darkness at 37 ° C for 2 hours, followed by dissolution of crystals of formazan DMSO and measuring the optical density at 590 nm using a plate reader. All MTT analyzes were performed in triplicate.

To assess neuritogenesis, the mean cell area was calculated using the ImageJ software. For this, the total area of SHSY-5Y cells stained with antibodies against β3-tubulin was determined and divided by the total number of nuclei labeled with DAPI dye using the color range parameters.

As a result, incubation with the conditioned medium of glial progenitors at a concentration of 5 μg / ml increases the number of living SH-SY5Y cells by 25.26 ± 4.5%. The addition of a secretome of neuronal precursors on the seventh day stimulates the growth of neurites in neuroblastoma cells, the area of which is 1.8 times the area of the group without therapy.

Thus, the addition of conditioned medium obtained from glial progenitors under hypoxic conditions of the SH-SY5Y cell line leads to a significant increase in survival and stimulation of neuritogenesis.

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Claims (3)

1. A method of obtaining a nootropic composition based on polypeptide complexes isolated from glial progenitor cells under heat shock conditions, including: taking a biopsy of the skin, obtaining a culture of fibroblasts, cryopreservation of a culture of fibroblasts, thawing of a culture of fibroblasts, adaptation of a culture of fibroblasts to xeno free conditions of fibroblasts, reprogramming for the production of induced pluripotent stem cells (iPSCs), culturing iPSCs, cryopreservation of iPSCs, thawing iPSCs, differentiation of iPSCs into glial progenitor cells, culturing glial progenitor cells under heat shock conditions, collecting conditioned medium and obtaining a protein-peptide complex, where, to simulate heat shock, glial precursors are cultured for an hour at 40 ° C in a growth medium containing DMEM / F12, 2% B27 additive, 20 ng / ml FGF-2, 1 μM purmorphamine, after which the medium is replaced with DMEM / F12 and cultured for 6 hours, after which the conditioned medium is collected and centrifuged at 300 rpm for 5 minutes, the supernatant is taken and concentrated 24 times using 3 kDa Amicon Ultra membranes, then the resulting concentrate is subjected to sterilizing filtration to obtain a nootropic composition. 2. Nootropic composition based on polypeptide complexes isolated from glial progenitor cells under heat shock conditions, characterized by the fact that it contains proteins and polypeptides with a molecular weight of 3 to 250 kDa, of which 65% have a molecular weight in the range from 10 up to 60 kDa, the content of heat shock proteins is not less than 40% of the total mass of proteins, brain neurotrophic factor (BDNF) is not less than 140 pg / ml and glial neurotrophic factor (GDNF) is not less than 60 pg / ml, vascular endothelial growth factor (VEGF) not less than 80 pg / ml, the total mass of the protein is not less than 1 mg / ml of the preparation, while the cell culture of glial progenitor cells from which the composition is obtained demonstrates the expression of astroglial markers S100b and GFAP and consists of 98 ± 2% S100b ⁺ - cells.

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