Effect Of Cerebrospinal Fluid On The Proliferation And Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells Derived Neural Stem Cells

Objective:To explore the effects of different concentrations cerebrospinal fluid(CSF)on the proliferation and differentiation of human umbilical cord mesenchymal stem cells(HUCMSCs)-derived neural stem cells(NSCs),to provide theoretical basis for NSCs transplantation and repair for neurodegenerative diseases.Methods:(1)HUC-MSCs were isolated and cultured from umbilical cords of healthy newborn infants in vitro.After subculture to P3 generation,NSCs were induced by orientation and their specific surface marker Nestin(neuroepithelial stem cell protein)were detected by flow cytometry for identification.(2)HUC-MSCs-derived NSCs were prepared into single-cell suspensions and uniformly inoculated into 96-well plates.Different concentrations of cerebrospinal fluid(0%,5%,10%,20%,30%,50%)were added and labeled for: 0% CSF group(blank control group),5% CSF group,10% CSF group,20% CSF group,30% CSF group,50% CSF group.After they cultured together for 72 h,the effects of different concentrations cerebrospinal fluid on the proliferation rate of HUC-MSCs-derived NSCs were detected by MTT colorimetry.(3)the well-grown HUC-MSCs derived NSCs after subculture were inoculated in 24-well plates to prepare cell climbing slides.After they co-cultured with the cerebrospinal fluid at different concentrations for 14 days,detect the neuronal marker(MAP-2)and astrocyte marker(GFAP)with immunofluorescent staining to observe the differentiation of HUC-MSCs derived NSCs into neurons and astrocytes.Results:(1)After co-culturing of HUC-MSCs derived NSCs with different concentrations of cerebrospinal fluid for 72 hours,the relative proliferation rates of the 5% CSF group,10% CSF group,20% CSF group,30% CSF group,and 50% CSF group are:(107±8.39)%,(131±15.44)%,(154±19.71)%,(178±26.46)%,(206±22.43)%,compared with the control group,except for the 5% cerebrospinal fluid group,10%,20%,30%,50 % Cerebrospinal fluid significantly increased the proliferation of HUC-MSCs derived NSCs after 72 h(P<0.01).(2)On the 14 th day of induction differentiation of HUCMSCs derived NSCs,MAP-2(+)neurons and GFAP(+)astrocytes were observed in the control group and each experimental group;The proportion of MAP-2(+)cells in the group(blank control group),5% CSF group,10% CSF group,20% CSF group,30% CSF group,and50% CSF group were:(16.01 ± 0.49)%,(16.30 ± 1.01)%,(16.39 ± 0.71)%,(16.55 ± 0.91)%,(25.06 ± 0.65)%,(29.38 ± 0.73)%;the proportion of GFAP(+)cells are:(6.80 ± 0.69)%,(7.01 ± 0.98)%,(7.14 ± 0.75)%,(7.75 ± 1.10)%,(12.55 ± 1.39)%,(17.21 ± 0.99)%.Compared with the control group,the proportion of HUC-MSCs-derived NSCs differentiated into neurons and astrocytes in the 30% and 50% CSF groups was significantly increased(P<0.01),while 5%,10%,and 20% CSF make no difference in the ratio of HUC-MSCs derived NSCs to neurons and astrocytes in the group(P> 0.05).Conclusion:(1)HUC-MSCs can be induced to NSCs in vitro and can further differentiate into neurons and astrocytes;(2)10%CSF promote the proliferation of HUC-MSCs derived NSCs in vitro.(3)30% CSF promote the differentiation of the HUC-MSCs derived NSCs in to the neurons and astrocytes;(4)HUC-MSCs derived NSCs are more likely to differentiate into neurons than astrocytes in 5%-50% low-medium concentration cerebrospinal fluid.