WO2018078407A1 - Procédé d'isolement de cellules progénitrices à partir de différents organes par destruction naturelle de matrice extracellulaire - Google Patents

Procédé d'isolement de cellules progénitrices à partir de différents organes par destruction naturelle de matrice extracellulaire Download PDF

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WO2018078407A1
WO2018078407A1 PCT/IB2016/001631 IB2016001631W WO2018078407A1 WO 2018078407 A1 WO2018078407 A1 WO 2018078407A1 IB 2016001631 W IB2016001631 W IB 2016001631W WO 2018078407 A1 WO2018078407 A1 WO 2018078407A1
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progenitor cells
fetal
cells
day
placenta
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WO2018078407A9 (fr
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Andrii KUKHARCHUK
Oleksandr KUKHARCHUK
Rohit Kulkarni
Abhijit BOPARDIKAR
Sunil POPHALE
Padma Priya Anand BASKARAN
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Reelabs Private Lmited, A Company Incorporated Under Provisions Of The Companies Act 1956
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Publication of WO2018078407A1 publication Critical patent/WO2018078407A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

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  • the present invention relates to the isolation of fetal progenitor cells from the organs of an aborted fetus after medical termination of a pregnancy, wherein said progenitor cells are available for subsequent therapeutic use.
  • BACKGROUND OF THE INVENTION [0002] Progenitor cells are already more specialised than a stem cells and are restricted to differentiate into tissue-specific or organ-specific cells. The most important difference between stem cells and progenitor cells is that progenitor cells have on their surface many regulatory proteins, which provide them with primary specialization.
  • the extracellular matrix is a collection of extracellular molecules secreted by cells that provides structural and biochemical support to the surrounding cells.
  • the ECM is mainly composed of an intricate interlocking mesh of fibrillar and non-fibrillar collagens, elastic fibers and glycosaminoglycan-containing non-collagenous glycoproteins (hyaluronan and proteoglycans). This tissue compartment provides structural support by maintaining organs and complex tissues. ECM of the fetus and the adult is significantly different. In the study by Coolen et al. (2010) most differences between fetal and adult skin were found in the expression pattern of ECM molecules. Both fibronectin and chondroitin sulfate were more abundantly present in the fetal dermis than in adult dermis, and elastin was not found in the
  • Matrix metalloproteinases is a group of /inc-dependent en/ymes. play significant roles in such remodeling. Specifically. M MP-2 and M MP-9. known as gelatinase A and B, respectively, are capable of degrading collagen type IV. elastin. and fibronectin and have been identified in the fetal membranes, decidua. and amniotic fluid.
  • MMPs plays a critical role in the invasive growth of the placenta.
  • MMP-2 localized to the amnion mesenchyme, chorion laeve trophoblast. decidua parietalis. and blood vessels in placenta vil li.
  • MM P-9 localized mainly to amnion epithelia.
  • FIG. 1 is a schematic that demonstrates the techniques of three open-loops fetal internal organs perfusion in situ. The fetus was rinsed with the 40% spirit solution, and placed onto a sterile surgical tray.
  • the entire procurement procedure was performed in a sterile environment with a laminar air flow unit providing European Union GMP class A (US class 100) air quality.
  • the abdominal and thoracic cavities accessed through a sternotomy and a midline laparotomy with bilateral subcostal lateral extensions.
  • the umbilical vein, aorta of left heart ventriculus and abdominal part of aorta cannulated with a 16- to 24-gauge cannula according to the size of the vessel, and the cannula ligated with silk.
  • FIG. 2 provides a bar graph showing proteolytic, colagenolytic and fibrinolytic activity in tissues of fetal intact internal organs.
  • FIG. 3 provides a bar graph showing comparison of the proteolytic and fibrinolitic activity in tissues of fetal intact internal organs and abortive placentas of the same fetuses.
  • Proteolitic activity of placenta tissue extracts was significantly higher than in tissues of fetal internal organs. Lysis of azoalbumin (782.3 ⁇ 24.69 ⁇ g/1 g tissue per 1 h) was 10.6 times higher than in suprarenal glands tissue and 47 times higher than in fetal heart (16.65 ⁇ 0.56 ⁇ g/1 g tissue per 1 h). Intensivity of azocazein lysis in placenta (753.7 ⁇ 16.99 g tissue per 1 h) was 11.5 times more than in suprarenal glands and 65.9 times higher than in tissue of fetal spleen (11.44 ⁇ 0.93 ⁇ g/1 g tissue per 1 h).
  • Collagenolytic activity also have maximal rate in placenta extract (405.3 ⁇ 19.71 ⁇ g/1 g tissue per 1 h) and was 5.6 and 49.4 times higher than in suprarenal glands and fetal kidney (8.21 ⁇ 0.26 ⁇ g/1 g tissue per 1 h) relatively.
  • FIG. 4 provides a bar graph showing total amount of progenitor cells, separated from fetal internal organs.
  • group A perfusion in situ by non-activated placenta extract
  • maximum rates of total cells were separated from fetal liver (40.0 ⁇ 5.36 ⁇ 10 6 per 1 g tissue), kidney (34.3 ⁇ 4.80 ⁇ 10 6 per 1 g tissue), spleen (32.9 ⁇ 5.03 ⁇ 10 6 per 1 g tissue) and lung (15.6 ⁇ 2.98 ⁇ 10 6 per 1 g tissue).
  • liver 44.3 ⁇ 6.00 ⁇ 10 6 per 1 g tissue
  • kidney 38.9 ⁇ 5.12 ⁇ 10 6 per 1 g tissue
  • spleen 38.2 ⁇ 5.67 ⁇ 10 6 per 1 g tissue
  • lung 21.1 ⁇ 4.13 ⁇ 10 6 per 1 g tissue
  • ileum 39.7 ⁇ 4.32 ⁇ 10 5 cells per 1 g tissue
  • suprarenal glands 43.4 ⁇ 4.95 ⁇ 10 5 cells per 1 g tissue
  • heart 9.1 ⁇ 1.88 ⁇ 10 5 cells per 1 g tissue
  • thymus 108.4 ⁇ 10.26 ⁇ 10 5 cells per 1 g tissue.
  • FIG. 5 provides a bar graph showing percent of viable progenitor cells, separated from internal organs of aborted fetuses. Analyses of viability of separation cells (tripan blue) shows that in group B percent of viable cells was significantly low: liver cells– 98.14 ⁇ 0.53% in group A vs 86.15 ⁇ 0.94% in group B; for kidney cells– 96.13 ⁇ 0.89% vs 85.02 ⁇ 1.26%; spleen cells– 97.41 ⁇ 0.75% vs 91.55 ⁇ 0.92%; lung cells– 93.82 ⁇ 1.05% vs 83.76 ⁇ 1.27%; ileum cells— 95.60 ⁇ 0.99% vs 88.14 ⁇ 0.86%; suprarenal glands cells– 98.15 ⁇ 0.66% vs 89.25 ⁇ 1.37%; heart cells– 92.57 ⁇ 1.16% vs 86.15 ⁇ 1.53%; thymus cells– 97.63 ⁇ 0.94% vs 90.10 ⁇ 1.19% relatively.
  • FIG. 6 provides Table 1 representing a design of single-dose toxicity studies. Single-dose Toxicity studies was carried out in 2 rodent species– mice and rats. In this study the same route of administration was used as intended for humans: intravenous fetal progenitor cells injections (rats). In addition, single-dose toxicity in cases of intra peritoneum (in mice) administration was also investigated. [0016] FIG. 7 (Table 2), and FIG. 8 (Table 3) provide the results of single-dose toxicity studies in rats after intravenous fetal progenitor cells injections. Animals were observed for 14 days after the fetal progenitor cells administration, and minimum lethal dose was not established. No mortality and signs of intoxication were observed.
  • FIG. 9 (Table 4) provides the results of haematological, coagulation, urinalysis, and blood biochemical studies evidences after intravenous fetal progenitor cells injections: all research parameters did not vary from control levels.
  • FIG. 10 (Table 5) provides the design of single-dose toxicity studies in mice after intra- peritoneum fetal progenitor cells injections described in the table.
  • FIG. 11 (Table 6), and FIG. 12 (Table 7) provides the results of single-dose toxicity studies in mice. Animals were observed for 14 days after the intra-peritoneum fetal progenitor cells administration, and minimum lethal dose was not established.
  • FIG. 13 (Table 8) provides the results of haematological, coagulation, urinalysis, and blood biochemical studies evidences that after intra-peritoneum fetal progenitor cells administration all research parameters do not vary from control parameters.
  • FIG. 14 (Table 9) provides the results of a study demonstrating the influence of human fetal progenitor cells transplantation on the quantity of leukocytes and leukocytes blood formula in Wistar rats.
  • FIG. 15 (Table 10) provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on the quantity of leukocytes and leukocytes blood formula in Wistar rats. Absolute quantity of eosinophils, basophils, neutrophils (metamyelocytes, band- nucleus and segmented-nucleus neutrophils), lymphocytes (small, moderate and large sizes) and monocytes similarly corresponding to parameters of control animal groups.
  • FIG. 15 (Table 10) provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on the quantity of leukocytes and leukocytes blood formula in Wistar rats. Absolute quantity of eosinophils, basophils, neutrophils (metamyelocytes, band- nucleus and segmented-nucleus neutrophils), lymphocytes (small, moderate and large sizes) and monocytes similarly corresponding to parameters of control animal groups.
  • FIG. 10 provides the results of a study
  • Table 11 provides the results of a study demonstrating the influence of human fetal progenitor cells transplantation on parameters of myelograms in Wistar rats.
  • parameters of myeologenesis and lymphopoiesis, index stimulation of neutrophilogenesis, percentage content of erythroblasts, pronormocytes and basophilic normocytes were undistinguished from the controls.
  • relative quantity of polychromatophilic and oxyphilic normocytes relatively grew to 39.3 and 61.3%. Besides, reduction of coefficient ratio of lymphoid and erythroid germs of the bone marrow to 18.4% were observed.
  • such parameters of percentage content in bone marrow as blasts promyelocytes, myelocytes, metamyelocytes, band-nucleus and segmented-nucleus neutrophils, eosinophils, basophils, monocytes, lymphocytes, plasmatic cells, megakaryocytes, erythroblasts, pronormocytes, basophilic normocytes and similarly index stimulation of neutrophilogenesis corresponded to controls.
  • Quantity of basophilic normocytes decreased to 30.1%.
  • Level of polychromatophilic and oxyphilic normocytes, on the contrary grew up to 1.4 and 2.0 times correspondingly.
  • FIG. 17 (Table 12) provides the results of a study demonstrating the influence of human fetal progenitor cells transplantation on erythron of Wistar rats.
  • animals of the first group observed increase content of hemoglobin in blood to 15.9% and reduction in erythrocyte sedimentation rate to 1.7 times.
  • Quantity of erythrocytes in peripheral blood and color parameter did not vary from the control.
  • bone marrow was noticed reduction in level of basophilic normocytes to 1.3 times and increase content of polychromatophilic normocytes to 1.2 times.
  • quantities of reticulocytes of bone marrow achieved the initial parameters on 60 th day of surveillance, but on the 120 th day content of reticulocytes were 35.2 % higher than the initial level.
  • FIG. 21 (Table 16) provides the results of a study demonstrating the influence of human fetal progenitor cells transplantation in relative mass of organs of the reproductive systems.
  • Table 18 provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on the activity of 3 ⁇ -hydroxyl- ⁇ 5 -steroid-dehydrogenase in testes of rats Wistar. Activity of 3 ⁇ -hydroxyl- ⁇ 5 -steroid-dehydrogenase after transplantation of fetal progenitor cells in intact rats tallied with the control parameters. Introduction of busulfan caused decrease in activity of 3 ⁇ -hydroxyl- ⁇ 5 -steroid-dehydrogenase to 26.8 and 28.2% correspondingly in recalculation on 1 mg of protein and 1 g of testes tissue.
  • FIG. 24 (Table 19) provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on concentration of spermatozoids in epididymises of the Wistar rats. Concentration of spermatozoids in epididymises of intact rats those were injected fetal progenitor cells matched with control.
  • Busulfan reduced the level of spermatozoids to 2.1 times. After introduction of busulfan and transplantation of fetal progenitor cells concentration of spermatozoids did not vary form the control parameters. Repeated intravenous introduction of fetal progenitor cells in immunosuppressed animals roused increase contents of spermatozoids in epididymises to 44.8% in comparison with control group.
  • FIG. 25 (Table 20) provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on index of spermatogenesis in testes of the rats Wistar.
  • FIG. 26 (Table 21) provides the results of a study demonstrating the influence of fetal progenitor cells transplantation on content of fructose in anterior part of prostates in the rats Wistar.
  • the method involve the use of fetal progenitor cells isolated from the organs of aborted fetuses after medical termination of pregnancy; the fetal progenitor cells are available for therapeutic use in accordance with organ-specific cell therapy and uses known to those skilled in the art.
  • the disclosed invention comprises the isolation of said progenitor cells comprising the use of natural collagenolytic, fibrinolytic and proteolytic activity; in certain embodiments, the collagenolytic, fibrinolytic and proteolytic extract are derived from the tissue of the abortive placenta.
  • Biological safety of fetal progenitor cells is provided by stringent control of bacteria, fungus and virus contamination in all stages of production– from procurement of anatomical material of aborted fetus which was destroyed as the result of medical termination of pregnancy till the preparation of cells suspension for study or treatment.
  • collection of fetal anatomical material is stringently controlled throuth numerous protocols. Purveyance of tissue of aborted fetuses was perfomed by Medical Termination of Pregnancy Rules, 2003. (G.S.R. 485(E)– In exercise of powers conferred by section 6 of the Medical Termination of Pregnancy Act, 1971 (34 of 1971), India).
  • the donor should be 18 years of age or above; the donor should be free from all the infectious diseases viz: HIV-1 & 2, HBV, HCV, CMV, VDRL; the duration of pregnancy should be between 18 to 20 weeks; the donor should undergo medical termination of pregnancy (MTP) and give her consent for the same independently of personnel who carries out the cell isolation.
  • MTP medical termination of pregnancy
  • Exclusion criteria for donor age of the pregnant women is less than 18 years; absence of“The Informed Consent for HIV Test”,“The Informed Consent for MTP” or“The Informed Consent for the collection of the abortive material” of the pregnant women; period of pregnancy is above 20 weeks; pregnant women is detected with infectious diseases including HIV 1/2, HCV, H ⁇ V, CMV, VDRL; known history for intrauterine fetal death; if the aborted material is collected as a result of spontaneous abortion; known for clear signs of congenital anomalies or infection in fetus.
  • a screening test for the donors is conducted as a second step.
  • MTP medical termination of pregnancy
  • The“Informed consent form for donating the aborted fetal and placenta/cord tissue for research” is provided by the hospital, where the pre-abortion tests and MTP is carried out. Procuring anatomical material is implemented only by obstetrician-gynaecologist and nurses of the medical institutions in which MTP is performed. [0037] The collection of fetal tissue material from aborted foetuses was performed in sterile condition, in accordance with the medical and health regulations mandated by the Indian goverment. Transportation of the fetal anatomical material (fetus and placenta) is performed in special cryo-bags, which eliminates the possibilities of microbial contamination while transportation.
  • Preliminary processing includes washing of anatomical material from blood and washed out solution taken for emergency microbial contamination analysis by the method of express endotoxin analysis.
  • Fetal placenta extract processing Large vessels were removed using blunt dissection and leaving only chorionic villous tissue, which was further dissected into 20 mg pieces. Tissues were extensively washed in sterile phosphate-buffered saline (PBS), and half of tissues were 5 min incubated in the presence of 25 ⁇ g/mL PGF2 ⁇ ( ⁇ nzaprost F, Chinoin Pharmaceutical and Chemical Works Private Co. Ltd., Hungary) for activation of proteolytic activity.
  • PBS sterile phosphate-buffered saline
  • Second half of tissue was not incubated, and used for study of natural proteolytic activity of placenta. Then all placenta villous tissues were homogenaized in tissue homogenaizer (Wheaton, USA). After centrifugation total volume of 25% supernatant was from 50 to 100 ml. Collagenase in human tissue is generally present in an inactivated form and thus needs to be activated. A 100-mL of supernatant was mixed with 80 mL of 125 mM borate buffer (pH 7.5) containing 10 mM CaCl 2 . Then, 10 mL of 43 ⁇ M trypsin was added and the solution was incubated at 37 ⁇ C for 10 min.
  • fetal progenitor cells isolation involves a three open-loops internal organs perfusion in situ (Fig.1). Blood from fetal vascular system was removed by perfusion of 1000 ml ice-cold D-PBS without calcium and magnesium. Remaining red blood cells in fetus vascular system were lysed by perfusion of ice-cold solution containing NH 4 Cl, KHCO 3 , and EDTA. The fetal vascular system was then perfused by ice-cold D-PBS without calcium and magnesium.
  • fetal vascular system was perfused by 25% fetal placenta tissue extract (placenta from same fetus) with CaCl2. Then fetal vascular system was perfused by D-PBS solution containing EDTA. Finally, fetus vascular system was perfused by ice-cold D-PBS without calcium and magnesium.
  • Soft and disintegrate encapsulated parenchymal organs (except lungs) were transferred into a 100-mm Petri dishes. The capsules were ruptured with forces and the mechanical disruption of the parenchymal organs was completed by scraping tissues with a sterile, disposable cell scrapers. This primary cells material was filtered through a sterile 500- ⁇ m nylon mesh.
  • Non-parenchymal organs and lungs were transferred into 100-mm Petri dishes. Organs were dissected into small fragments and transferred into tissue homogenizer, and minced into indiscrete mass. Cells were washed out from homogenizer walls and piston with Hanks’ solution into graduated test tubes passing them through blood transfusion filter and then through needles of more and more narrower diameter. After centrifugation the cells pellet was washed 2-4 times and transferred into cryo-vials after adding cryoprotector (DMSO, 5% final concentration).
  • cryoprotector DMSO, 5% final concentration
  • Biosafety control A portion of harvested cells were directed to biosafety lab and microbiological lab (the ready cells suspensions were studied for bacterial sterility, contamination of viruses, fungus and transmission infections: HIV1/HIV2, HbsAg, H ⁇ V, HBV, HSV 1/2, CMV, Treponema pallidum, Toxoplasma gondii, Micoplasma, Ureaplasma, Chlamidii, EBV by means of polymerase chain reaction). Cell viability was determined with Trypan Blue. [0043] Passportization (analytical characterisation) of fetal progenitor cells suspensions.
  • Progenitor cells passport contain: result of screening test for donors of anatomical material (ELISA): HIV 1, HIV 2, HBV, HCV, CMV, HTLV, VDRL; Cell Test-Control (PCR): HIV 1, HIV 2, HBV, HCV, CMV, HTLV, HSV1 &2, EBV, Treponema pallidum, Mycoplasma hominis, Ureaplasma sp., Chlamydia all species, Toxoplasma gondii; Microbiologist Control Sterility Testing: Aerobic m/o, Anaerobic m/o, Fungus; Gestation Period of the Human Fetus: 18-20 weaks; Progenitor cells types: liver, brain, spinal cord, gastric, intestinal, lung, kidney, skin, bone, oculus, heart, thymus, spleen, pancreas, muscle, vessels; Progenitor cells suspension quantity & quality: total cells amount per 1 ml, live-cell
  • progenitor cells suspensions are ready for use: liver progenitor cells, intestinal progenitor cells, lung progenitor cells, kidney progenitor cells, heart progenitor cells, suprarenal glands progenitor cells, thymus progenitor cells, and spleen progenitor cells.
  • proteolytic and colagenolitic activity of fetal organs tissue and fetal placenta extract were investigated.
  • Proteolytic and colagenolitic activities were studied by method of intensivity azoalbumin, azocazein and azocollagene lysis estimation.
  • Tissue fibrinolytic activity was investigated by azofibrin lysis.
  • epsilon-aminocapronic acid For analysis of non-enzymatic fibrinolysis epsilon-aminocapronic acid was used. As shown in Fig.2, maximum levels of azoalbumin, azocazein and azocollagen lysis were in tissue of fetal suprarenal glands. High fibrinolytic activity was observed in fetal liver. Proteolitic activity of placenta tissue extract was significally higher than in tissues of fetal internal organs (Fig. 3). Lysis of azoalbumin was 11 times higher than in suprarenal glands tissue and 47 times higher than in fetal heart. Intensivity of azocazein lysis in placenta was 12 times more than in suprarenal glands and 66 times higher than in tissue of fetal spleen.
  • Collagenolytic activity also have maximum rate in placenta extract and was 6 and 49 times higher than in suprarenal glands and fetal kidney relatively.
  • fetal placenta levels of total fibrinolytic activity non-enzymatic and enzymatic fibrinolysis were 12, 5, 17 times more than in suprarenal glands and 99, 42, 152 times higher than in tissue of fetal spleen correspondingly.
  • the natural proteolytic activity of the placenta is enough for the soft destruction of the immature extracellular matrix of the fetus organs in order to isolate progenitor cells.
  • Total amount of progenitor cells, separated from fetal internal organs (Fig. 4), in group A (perfusion in situ by natural proteolytic activity of the placenta extract) was maximum in liver, kidney, spleen and lung. From fetal ileum we separated 2.9–4.2 millions cells per 1 g tissue, suprarenal glands– 3.0–5.0, heart– 0.4–1.2, thymus– 8.1–12.2 millions cells per 1 g tissue.
  • Rats Wistar First group (group 1)– 14 rats (7 males, 7 females).
  • Characteristics of fetal progenitor cells Gestation term– 18-20 weeks; total quantity of cells– 31.05 ⁇ 10 6 /rat; quantity of spleen progenitor cells– 8.00 ⁇ 10 6 /rat; quantity of heart progenitor cells– 2.50 ⁇ 10 5 /rat; quantity of lung progenitor cells– 4.00 ⁇ 10 6 /rat; quantity of kidney progenitor cells– 8.00 ⁇ 10 6 /rat; quantity of suprarenal glands progenitor cells– 2.50 ⁇ 10 5 /rat; quantity of ileum progenitor cells– 3.00 ⁇ 10 5 /rat; quantity of thymus progenitor cells– 2.50 ⁇ 10 5 /rat; quantity of liver progenitor cells– 10.00 ⁇ 10 6 /rat.
  • Control group Intravenous 0.9% sodium chloride injection
  • 14 animals 7 males, 7 females. All investigation (analysis of peripheral blood and myelograms) were conducted after 28 days of subsequent transplantation fetal progenitor cells.
  • quantity of leukocytes in peripheral blood and parameters of leukocytes blood formula were undistinguished from the control (Fig. 14).
  • such parameters of percentage content in bone marrow as blasts promyelocytes, myelocytes, metamyelocytes, band-nucleus and segmented- nucleus neutrophils, eosinophils, basophils, monocytes, lymphocytes, plasmatic cells, megakaryocytes, erythroblasts, pronormocytes, basophilic normocytes and similarly index stimulation of neutrophilogenesis corresponded to controls.
  • Quantity of basophilic normocytes decreased to 30%.
  • Level of polychromatophilic and oxyphilic normocytes, on the contrary grew to 1.4 and 2.0 times correspondingly.
  • Characteristics of fetal progenitor cells Gestation term– 18-20 weeks; total quantity of cells– 134.20 ⁇ 10 6 /rabbit; quantity of spleen progenitor cells– 32.00 ⁇ 10 6 /rabbit; quantity of lung progenitor cells– 16.00 ⁇ 10 6 /rabbit; quantity of heart progenitor cells– 10.00 ⁇ 10 5 /rabbit; quantity of kidney progenitor cells– 32.00 ⁇ 10 6 /rabbit; quantity of suprarenal glands progenitor cells– 10.00 ⁇ 10 5 /rabbit; quantity of ileum progenitor cells– 12.00 ⁇ 10 5 /rabbit; quantity of thymus progenitor cells– 10.00 ⁇ 10 5 /rabbit; quantity of liver progenitor cells– 40.00 ⁇ 10 6 /rabbit.
  • Control group– 15 animals (8 males, 7 females): busulfan immunosuppressed rabbits, those were injected 0.9% sodium chloride solution instead of fetal progenitor cells.
  • rabbits 1.5 ml of blood were collected from right or left vena auricularis with the help of heparinized syringe. Bone marrow collected from femur by the washing method under the ether anesthesia.
  • quantities of reticulocytes of bone marrow achieved the initial parameters on 60 th day of surveillance, but on the 120 th day content of reticulocytes were 35% higher than the initial level.
  • band-nucleus and segmented-nucleus neutrophils corresponded to the initial parameters.
  • Relative quantity of basophils reduced only on 120 th day of surveillance.
  • On the 6 th and 18 th day noticed 40% decrease level of eosinophils in blood, but in remaining period of surveillance this parameter did not vary from the initial data.
  • Relative contents of lymphocytes in peripheral blood on 6 th day reduced to 35%, on 12 th day– to 26%, on 18 th day– to 28%, on 60 th day– to 18%.
  • On 120 th day quantity of leukocytes in blood did not vary from the initial level.
  • Carcinogenicity of fetal progenitor cells was studied by intravenous and ectopic introduction of cells to immunocompromized (busulphan, 1 mg per 1 kg body mass, once in a day, course of 7 days) rats, rabbits, guinea pigs and mice.
  • the cells were provided to the animals as follow: intradermally, subcutaneously, intramuscularly, in anterior chamber of eye, under the capsule of kidneys, in omentum, in liver tissue, in spleen tissue, in lung tissue, in the wall of small intestine, in wall of large intestine, in the wall of the stomach, in sternum.
  • fetal progenitor cells comprised the following: Gestation term– 18-20 weeks; total quantity of cells– 62.10 ⁇ 10 6 /rat; quantity of spleen progenitor cells– 16.00 ⁇ 10 6 /rat; quantity of heart progenitor cells– 5.00 ⁇ 10 5 /rat; quantity of lung progenitor cells– 8.00 ⁇ 10 6 /rat; quantity of kidney progenitor cells– 16.00 ⁇ 10 6 /rat; quantity of suprarenal glands progenitor cells– 5.00 ⁇ 10 5 /rat; quantity of ileum progenitor cells– 6.00 ⁇ 10 5 /rat; quantity of thymus progenitor cells– 5.00 ⁇ 10 5 /rat; quantity of liver progenitor cells– 20.00 ⁇ 10 6 /rat.
  • Fetal progenitor cells were injected via syringe in jugular vein after venal section under anesthesia (nembutal, 40 mg per 1 kg body mass, intra-peritoneal).
  • Busulfan injected intra-peritoneal, dose of 1 mg per kg body mass, once in a day during the course of 7 days; the total dose of busulfan consisted of 7 mg per kg body mass.
  • the mortality after injecting busulfan consisted of 53%.
  • Ventral and anterior parts of prostate glands, testes and epididymises were isolated after mortification of animals under ether anesthesia. The isolated organs were weighed in electronic weighing balance (Sartorius).
  • Busulfan shows expressed toxic effect in the reproductive system of the Wistar line male rats: Under the influence of this immunosuppressor mass of testes decreased to 2.0 times, concentration of testosterone in blood plasma decreased to 4.1 times, content of biological active luteinizing hormone in blood decreased to 2.0 times, activity of 3 ⁇ - hydroxyl- ⁇ 5 -steroid-dehydrogenase reduces to 27%, concentration of spermatozoids in epididymises decreased to 2.1 times, index of spermatogenesis decreased to 2.3 times, content of fructose in the anterior part of prostates decreased to 26%.
  • MMP activity may be required during development and normal physiology in several ways: (1) to degrade ECM molecules and allow cell migration; (2) to alter the ECM micro-environment and result in alteration in cellular behavior; (3) to modulate the activity of biologically active molecules by direct cleavage, release from bound stores, or the modulating of the activity of their inhibitors [Vu1 T.H, Werb Z. (2000) Matrix metalloproteinases: effectors of development and normal physiology Gen Develop 14: 2123–2133]. [0057] In placenta proteolysis is associated mainly with the invasive growth that proposes more intensive degradation of mature extracellular matrix components of the uterus endometrium.
  • MMPs expressed at high mRNA levels in the first-trimester trophoblast of the human placenta Hiden U. et al. (2008) Diabetes].
  • the prominent expression of MT1-MMP early in gestation suggests a major role in processes involved in early placental development. This notion is further supported by its predominant presence in the HLA-G–expressing trophoblast subpopulation, which represents the invasive extravillous trophoblast.
  • the high MT1-MMP expression in the HLA-G–positive trophoblasts is in accordance with results of in situ hybridization in first- trimester placental tissue [Bjorn SF, Hastrup N, Lund LR, Dano K, Larsen JF, Pyke C: (1997) Mol Hum Reprod].
  • MMP-9 was localized mainly to amnion epithelia, chorion laeve trophoblast, decidua parietalis, and placenta syncytiotrophoblasts. Separate cell culture of purified placenta trophoblast cells showed that placenta syncytiotrophoblast and amnion epithelial cells exclusively produced MMP-9; chorion trophoblast cells secreted both MMP-2 and MMP-9. An increase in MMP-9 expression may contribute to degradation of the ECM in the placenta.
  • MMPs to involve in multiple steps of trophoblast invasion penetration of the endometrial epithelium, rupture of the endometrial basement membrane, infiltration through the endo- and myometrium, penetration and conversion of the maternal spiral arteries, and finally, cessation of invasion [Xu P., Alfaidy N., Challis J.R.G. (2002) J Clin Endocrinol Metab]. Xu P. et al.
  • MMP-2 is the predominant form of type IV collagenases while MMP-9 accounts for a minor amount
  • MMP-9 accounts for a minor amount
  • Fertil Steril after the blastocysts implant into the endometrium and before placentation is complete in the first trimester, human trophoblasts produce both MMP-2 and MMP-9 [Polette M, Nawrocki B, Pintiaux A, Massenat C, Maquoi E, Volders L, Schaaps JP, Birembaut P, Foidart JM. (1994) Lab Invest; Shimonovitz S, Hurwitz A, Dushnik M, Anteby E, Geva-Eldar T, Yagel S.
  • PA activity in vascular and extracellular spaces is modulated by PA inhibitors (PAIs). PA-PAI interactions modulate trophoblast invasion in vivo and control fibrinolysis within the intervillous spaces of the placenta.
  • PAI-1 and PAI-2 mRNA and protein are produced by cultured cytotrophoblasts.
  • PAI-2 was localized by immunocytochemistry to villous syncytiotrophoblasts whereas PAI-1 was present primarily in invasive trophoblasts of implantation sites.
  • abortive placental extract has a high proteolytic, fibrinolytic and collagenolytic activity, it can be used for the purpose of degradation of the extracellular matrix of fetal organs to isolate progenitor cells.
  • Consequent step was to study the amount of cells from different organs of the fetus can be obtained by perfusion of the autologous placenta tissue extract.
  • Additional yields include the following: from fetal ileum 36.8 ⁇ 3.75 ⁇ 10 5 cells per 1 g tissue, suprarenal glands– 40.8 ⁇ 5.19 ⁇ 10 5 cells per 1 g tissue, heart– 7.2 ⁇ 1.60 ⁇ 10 5 cells per 1 g tissue, thymus– 98.2 ⁇ 9.31 ⁇ 10 5 cells per 1 g tissue. Amount of separated cells increased after fetal placenta incubation in presence PGF2 ⁇ , but viability of these cells dramatically decreased. (Accordingly, PGF2 ⁇ -treated placenta extracts cells were not included in final study of fetal progenitor cells biosafety).
  • liver weight from 10 till 15 g)– 40-60 ⁇ 10 7
  • kidney weight from 1.5 till 3.0 g)– 50-100 ⁇ 10 6
  • spleen weight from 0.2 till 0.4 g)– 7-13 ⁇ 10 6
  • lung weight from 6 till 9 g)– 90-140 ⁇ 10 6
  • heart weight from 1.5 till 2.5 g)– 1-2 ⁇ 10 6
  • thymus weight from 0.3 till 0.6 g)– 3-6 ⁇ 10 6 , which is sufficient for organ-specific cell therapy without prior multiplication of progenitor cell.
  • progenitor cells for cell therapy requires, first of all, an evaluation of their safety.
  • the single-dose data demonstrates the absence of fetal progenitor cells toxic effects after their intravenous introduction (rat) and intra-peritoneal injection (mice). In additional, no toxic effect were observed in blood system of Wistar rats.
  • Weak expressive activation of erythropoiesis was observed in bone marrow.
  • peripheral blood insignificantly increases level of hemoglobin without reliable changes in quantity of erythrocytes, and decreased erythrocytes sedimentation rate were observed.
  • moderate expressive activation of erythropoiesis was observed in bone marrow.
  • Busulfan shows expressed toxic effect in the reproductive system of the Wistar line male rats: Under the influence of this immunosuppressor mass of testes decreases to 2.0 times, concentration of testosterone in blood plasma– to 4.1 times, content of biological active luteinizing hormone in blood– to 2.0 times, activity of 3 ⁇ - hydroxyl- ⁇ 5 -steroid-dehydrogenase reduces to 27%, concentration of spermatozoids in epididymises– to 2.1 times, index of spermatogenesis– to 2.3 times, content of fructose in the anterior part of prostates – to 26%.

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Abstract

La présente invention concerne de nouveaux procédés comprenant l'isolement de cellules progénitrices fœtales viables à partir d'organes d'un fœtus avorté. L'invention comprend l'utilisation de l'activité protéolytique, collagénolytique et fibrinolytique naturelle d'un extrait de tissu placentaire abortif autologue.
PCT/IB2016/001631 2016-10-29 2016-10-29 Procédé d'isolement de cellules progénitrices à partir de différents organes par destruction naturelle de matrice extracellulaire WO2018078407A1 (fr)

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Citations (3)

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US20060182724A1 (en) * 2005-02-15 2006-08-17 Riordan Neil H Method for expansion of stem cells
US20100144027A1 (en) * 2007-02-16 2010-06-10 Lee Jin-Pyo Placental protein having a regulating action on proteolytic activity and its related gene
CN102146359A (zh) * 2011-01-13 2011-08-10 王泰华 从胎盘中提取原始间充质干细胞及无血清扩增的方法

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Publication number Priority date Publication date Assignee Title
US20060182724A1 (en) * 2005-02-15 2006-08-17 Riordan Neil H Method for expansion of stem cells
US20100144027A1 (en) * 2007-02-16 2010-06-10 Lee Jin-Pyo Placental protein having a regulating action on proteolytic activity and its related gene
CN102146359A (zh) * 2011-01-13 2011-08-10 王泰华 从胎盘中提取原始间充质干细胞及无血清扩增的方法

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Title
POTDAR, PD: "Development and Molecular Characterization of Human Placental Mesenchymal Stem Cells from Human Aborted Fetal Tissue as a Model to Study Mechanism of Spontaneous Abortion", ADVANCES IN STEM CELLS, vol. 2014, no. 685337, 19 February 2014 (2014-02-19), pages 1 - 17, XP055479721 *
SHARMA, K ET AL.: "Immunoglobulin Isotype Isolated from Human Placental Extract does not Interfere in Complement-Mediated Bacterial Opsonization within the Wound Milieu", FEBS OPEN BIO., vol. 5, 18 April 2015 (2015-04-18), pages 369 - 377, XP055479722 *

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