Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages

BACKGROUND Mesenchymal stem cells(MSCs)have been extensively studied for therapeutic potential,due to their regenerative and immunomodulatory properties.Serial passage and stress factors may affect the biological characteristics of MSCs,but the details of these effects have not been recognized yet.AIM To investigate the effects of stress factors(high glucose and severe hypoxia)on the biological characteristics of MSCs at different passages,in order to optimize the therapeutic applications of MSCs.METHODS In this study,we investigated the impact of two stress conditions;severe hypoxia and high glucose on human adipose-tissue derived MSCs(hAD-MSCs)at passages 6(P6),P8,and P10.Proliferation,senescence and apoptosis were evaluated measuring WST-1,senescence-associated beta-galactosidase,and annexin V,respectively.RESULTS Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control,while the extent of senescence did not change significantly under stress conditions.At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6.Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage,while no change in apoptosis was observed.On the other hand,MSCs cultured under hypoxia showed decreased senescence,increased apoptosis and no significant change in proliferation when compared to the same conditions at P8.CONCLUSION These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages,and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.

Character of viral clones in serial passageof a nuclear polyhedrosis virus in vitro

In this paper,the character of viral clones from early and late passages after serial passages of Trichoplusia in single nuclear polyhedrosis virus in a Tn SB1-4 cell line is described.It demonstrated that no significant difference was observed in the infectivity of the cell culture supernatants of various passages to the cell line.The number of polyhedra produced in a cell and infechvity of polyhedra to T.ni larvae declined strikingly with the increase of passages.The polyhedra without vinons begun to increase from passage to passage.The result of restriction enzyme digestion showed that the DNA restriction fragments of the clones were different from wild virus DNA,although they came from a homogeneous viral DNA.The mutation of viral DNA resulted in the increase of noninfectious polyhedra without vinous and in the increase of the number of polyhedra produced in cell line as wen as virulence of the polyhydrosis inclusion bodys to T.ni larvae after prolonged passages of Tn SNPV in the cell culture.

A Virulent PEDV Strain FJzz1 with Genomic Mutations and Deletions at the High Passage Level Was Attenuated in Piglets via Serial Passage In Vitro

Highly virulent porcine epidemic diarrhea virus(PEDV)strains re-emerged and circulated in China at the end of 2010,causing significant economic losses in the pork industry worldwide.To understand the genetic dynamics of PEDV during its passage in vitro,the PEDV G2 strain FJzzl was serially propagated in Vero cells for up to 200 passages.The susceptibility and adaptability of the FJzzl strain increased gradually as it was serially passaged in vitro.Sequence analysis revealed that amino acid(aa)changes were mainly concentrated in the S glycoprotein,which accounted for 72.22%-85.71%of all aa changes.A continuous aa deletion(^(55)I^(56)G^(57)E→^(55)K^(56)Δ^(57)Δ)occurred in the N-terminal domain of S1(Sl-NTD).To examine how the aa changes affected its virulence,FJzzl-F20 and FJzzl-F200 were selected to simultaneously evaluate their pathogenicity in suckling piglets.All the piglets in the FJzzl-F20-infected group showed typical diarrhea at 24 h postinfection,and the piglets died successively by 48 h postinfection.However,the clinical signs of the piglets in the FJzzl-F200-infected group were significantly weaker,and no deaths occurred.The FJzzl-F200-infected group also showed a lower level of fecal viral shedding and lower viral loads in the intestinal tissues,and no obvious histopathological lesions.TypeⅠandⅢinterferon were induced in the FJzzl-F200 infection group,together with pro-inflammatory cytokines,such as TNF-α,IL-1βand IL-8.These results indicate that the identified genetic changes may contribute to the attenuation of FJzzl strain,and the attenuated FJzzl-F200 may have the potential for developing PEDV live-attenuated vaccines.

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019,Severe Acute Respiratory Syndrome Coronavirus 2(SARS-Co V-2)keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner.To gain deeper insight into mutation frequency and dynamics,we isolated ten ancestral strains of SARS-Co V-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-q PCR and whole genome sequencing.Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-Co V-2 in vitro.Our results identified a series of adaptive genetic changes,ranging from unique convergent substitutional mutations and hitherto undescribed insertions.The region coding for spike proved to be a mutational hotspot,evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501.We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-Co V-2 in the adaptation to cell culture.The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-Co V-2 strains.The observed genetic changes of SARS-Co V-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants,along the path of increasing potency of the strain.Competition among a high number of quasi-species in the SARS-Co V-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.