scholarly journals More Human BM-MSC With Similar Subpopulation Composition and Functional Characteristics Can Be Produced With a GMP-Compatible Fabric Filter System Compared to Density Gradient Technique

2021 ◽  
Vol 9 ◽  
Author(s):  
Gabriele Spohn ◽  
Anne-Sophie Witte ◽  
Anja Kretschmer ◽  
Erhard Seifried ◽  
Richard Schäfer
Keyword(s):  
Growth Factor ◽  
Density Gradient ◽  
Trophic Factors ◽  
Woven Fabric ◽  
Functional Characteristics ◽  
Filter System ◽  
Fabric Filter ◽  
Trilineage Differentiation ◽  
Immunomodulatory Potential

BackgroundMesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)–compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.MethodsHuman BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.ResultsThe CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 109 vs. 1.45 × 108). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a+CD119+CD146+ as most common phenotype group, and CD140a+CD119+CD146+MSCA-1–CD106–CD271– and CD140a+CD119+CD146–MSCA-1–CD106–CD271– as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.ConclusionThe isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.

scholarly journals Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo

2019 ◽  
Vol 20 (18) ◽  
pp. 4486 ◽  
Author(s):  
Sabrina Petralla ◽  
Luis Emiliano Peña-Altamira ◽  
Eleonora Poeta ◽  
Francesca Massenzio ◽  
Marco Virgili ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Cell Model ◽  
Precursor Cells ◽  
Trophic Factors ◽  
Psychomotor Development ◽  
Oligodendrocyte Precursor ◽  
Aspartate Glutamate Carrier

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate–aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor βs (TGFβs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.

180 Increasing GfrA1-Positive Spermatogonial Stem Cell Population of Goat

2018 ◽  
Vol 30 (1) ◽  
pp. 230 ◽  
Author(s):  
V. Sharma ◽  
S. Saini ◽  
B. Aneja ◽  
A. Kumar ◽  
A. Thakur ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Growth Factor ◽  
In Vitro Culture ◽  
Density Gradient ◽  
Stem Cell Population ◽  
Seminiferous Tubules ◽  
Rt Pcr ◽  
Pluripotency Markers ◽  
Percoll Density Gradient

Spermatogonial stem cells (SSC) form the basis of spermatogenesis and continuous fertility in male. Their meagre population in the testis is a hindrance in the in vitro study of biological activity of these cells. The objective of the present study was to isolate and characterise goat SSC and increase their number during in vitro culture by different methods. Two goat testes (3 to 4 months of age) were collected from the slaughterhouse and transported to the laboratory. The testes were washed and seminiferous tubules were collected and minced in the laminar flow hood. The seminiferous tubules were washed twice with PBS to remove spermatozoa and subjected to double enzymatic digestion (collagenase, 1 mg mL−1, hyaluronidase, 1 mg mL−1, trypsin, 0.05%, and DNaseI, 10 µg mL−1 for 45 min and second digestion with same set of enzymes except trypsin for 30 min). The isolated cells were filtered sequentially through nylon mesh filters of pore size 70 and 40 µm. The cells were plated on DSA-lectin coated dishes for 4 h and the unattached cells were cultured on a Sertoli cell feeder layer prepared by treating with mitomycin-C for 3 h. The cells were cultured in DMEM/F-12 supplemented with human recombinant growth factors (glial cell-derived neurotrpic factor, 10 ng mL−1, fibroblast growth factor FGF, 10 ng mL−1, epidermal growth factor, 20 ng mL−1), 10% fetal bovine serum, and antibiotics. The expression of pluripotency markers (Oct4, Nanog, Sox2) and SSC-specific markers (Thy1, GfrA1, and Uchl1) in the SSC colonies was determined by RT-PCR and immunostaining, after in vitro culture of 3 weeks. The SSC population was enhanced by differential plating, Percoll density gradient (on Day 1) and SSC passaging (by passaging SSC colonies on Day 20). The cells were tagged with GfrA1 antibody and their population was tested by flow cytometry. The SSC colonies started appearing after 7 days and continued to grow in size and number until 3 weeks. The SSC colonies were positive for the pluripotency markers Oct4, Nanog, and Sox2 by RT-PCR and immunostaining. The SSC were also positive for the SSC-specific markers Thy1, GfrA1, and Uchl1 by RT-PCR and immunostaining. Flow cytometry showed that the GfrA1-positive population in the SSC enriched by the differential plating was 11.23%, Percoll density gradient was 23.57%, and by passaging of SSC colonies, after picking and trypsinising with 0.05% trypsin, was 91.23%. In vitro culture of the SSC enriched by these methods also revealed that the number of SSC colonies appearing in the cells enriched by passaging was higher than the other methods. From the results of present study, we conclude that SSC are positive for markers of pluripotency and SSC-specific markers. The SSC population can be enhanced to a very high level following SSC passaging, which is an inexpensive method and does not require expensive instruments like fluorescence- or magnetic-activated cell sorting.

Keratinocyte Growth Factor (KGF) beeinflusst das Wachstum von Hepatom-Zellen in vitro

2005 ◽  
Vol 43 (05) ◽  
Author(s):  
M Mühlbauer ◽  
T Amann ◽  
J Schölmerich ◽  
A Bosserhoff ◽  
C Hellerbrand
Keyword(s):  
Growth Factor ◽  
Keratinocyte Growth Factor

Neuroprotective and anti-amnestic effects of a combination therapy in a model of photochemical ischemic damage in the prefrontal cortex

2018 ◽  
pp. 39-45
Author(s):  
Ф.М. Шакова ◽  
Т.И. Калинина ◽  
М.В. Гуляев ◽  
Г.А. Романова
Keyword(s):  
Prefrontal Cortex ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Combination Therapy ◽  
Neuroprotective Effect ◽  
Regulatory Protein ◽  
Human Growth ◽  
Neuroprotective Effects ◽  
Nerve Growth

Цель исследования - изучение влияния комбинированной терапии (мутантные молекулы эритропоэтина (EPO) и дипептидный миметик фактора роста нервов ГК-2H) на воспроизведение условного рефлекса пассивного избегания (УРПИ) и объем поражения коры мозга у крыс с двусторонним ишемическим повреждением префронтальной коры. Методика. Мутантные молекулы EPO (MЕРО-TR и MЕPО-Fc) с значительно редуцированной эритропоэтической и выраженной цитопротекторной активностью созданы методом генной инженерии. Используемый миметик фактора роста нервов человека, эндогенного регуляторного белка, в экспериментах in vitro проявлял отчетливые нейропротективные свойства. Двустороннюю фокальную ишемию префронтальной коры головного мозга крыс создавали методом фотохимического тромбоза. Выработку и оценку УРПИ проводили по стандартной методике. Объем повреждения мозга оценивался при помощи МРТ. MEPO-TR и MEPO-Fc (50 мкг/кг) вводили интраназально однократно через 1 ч после фототромбоза, ГК-2Н (1 мг/кг) - внутрибрюшинно через 4 ч после фототромбоза и далее в течение 4 послеоперационных суток. Результаты. Выявлено статистически значимое сохранение выработанного до ишемии УРПИ, а также значимое снижение объема повреждения коры при комплексной терапии. Полученные данные свидетельствуют об антиамнестическом и нейропротекторном эффектах примененной комбинированной терапии, которые наиболее отчетливо выражены в дозах: МEPO-Fc (50 мкг/кг) и ГК-2Н (1 мг/кг). Заключение. Подтвержден нейропротекторный эффект и усиление антиамнестического эффекта при сочетанном применении мутантных производных эритропоэтина - MEPO-TR и MEPO-Fc и дипептидного миметика фактора роста нервов человека ГК-2H. The aim of this study was to investigate the effect of combination therapy, including mutant erythropoietin molecules (EPO) and a dipeptide mimetic of the nerve growth factor, GK-2H, on the conditioned passive avoidance (PA) reflex and the volume of injury induced by bilateral ischemia of the prefrontal cortex in rats. Using the method of genetic engineering the mutant molecules of EPO, MERO-TR and MEPO-Fc, with strongly reduced erythropoietic and pronounced cytoprotective activity were created. The used human nerve growth factor mimetic, an endogenous regulatory protein based on the b-bend of loop 4, which is a dimeric substituted dipeptide of bis- (N-monosuccinyl-glycyl-lysine) hexamethylenediamine, GK-2 human (GK-2H), has proven neuroprotective in in vitro experiments. Methods. Bilateral focal ischemic infarction was modeled in the rat prefrontal cortex by photochemically induced thrombosis. The PA test was performed according to a standard method. Volume of brain injury was estimated using MRI. MEPO-TR, and MEPO-Fc (50 mg/kg, intranasally) were administered once, one hour after the injury. GK-2Н (1 mg/kg, i.p.) was injected four hours after the injury and then for next four days. Results. The study showed that the complex therapy provided statistically significant retention of the PA reflex developed prior to ischemia and a significant decrease in the volume of injury. The anti-amnestic and neuroprotective effects of combination therapy were most pronounced at doses of MEPO-Fc 50 mg/kg and GK-2H 1 mg/kg. Conclusion. This study has confirmed the neuroprotective effect and enhancement of the anti-amnestic effect exerted by the combination of mutant erythropoietin derivatives, MEPO-TR and MEPO-Fc, and the dipeptide mimetic of human growth factor GK-2H.

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