The Evaluation Of Human Umbilical Cord Blood CD133~+ Cells In The Construction Of Humanized Mouse Model And The Effect Of Berberine On Its Expansion In Vitro

BackgroundThe humanized mouse model is considered to be the best tool for basic immune research in the process of disease development and preclinical drug evaluation.However,the construction effect of this model is affected by many factors such as cell source,transplantation route,immune-deficient mouse strain and pretreatment method.At present,humanized mouse models are usually established by injecting human umbilical cord blood(UCB)CD34~+hematopoietic stem cells(HSCs)into the radiation-treated neonatal immune-deficient mice through the tail vein.However,it has been reported that human UCB-CD133~+HSCs are better than CD34~+HSCs in the number and quality of differentiation after transplantation.Intramedullary injection(IBMI)can more effectively transplant HSCs.NOG(NOD Shi-SCID IL-2Rγcnull)mice are the best receptor for HSCs.In addition,NOG mice are sensitive to radiation and radiation treatment requires special equipment and personnel.Busulfan,a pretreatment drug commonly used in clinical transplantation,is an ideal alternative.The effect of humanized mouse immune system reconstruction depends on the number of HSCs transplanted,and more HSCs can often achieve higher reconstruction levels.However,the number of HSCs that can be separated in a single UCB is limited,and a large number of humanized mouse models cannot be constructed.If humanized mice constructed by multiple UCB-HSCs are used in the same experiment,it is difficult to control individual differences impact of results.Therefore,how to obtain a large amount of homogeneous human HSCs through in vitro amplification is an urgent problem to be solved.ObjectiveTransplant human UCB-CD133~+HSCs into NOG mice pretreated with Busulfan by bone marrow injection to construct a humanized mouse model with human immune system;Detect the effect of berberine on the in vitro expansion of human UCB-CD133~+HSCs.Method1.Human UCB-CD133~+HSCs were transplanted into the bone marrow cavity of NOG mice pretreated with Busulfan to construct a humanized mouse model of the immune system and evaluate the construction effect;1.1 Use Ficoll density gradient centrifugation to separate mononuclear cells(MNCs)from healthy pregnant women UCB,and then use magnetic beads sorting method to sort CD133~+HSCs from MNCs.1.2 Use Flow cytometry to detect the percentage of CD133~+cells in MNCs and the purity of CD133~+cells sorted by magnetic beads.1.3 Busulfan pretreatment/normal saline pretreatment(control)NOG mice.CD133~+HSCs were transplanted into the bone marrow cavity of mice after 24 hours.Observe and record the behavioral changes and survival of mice every week after transplantation.1.4 Collect the peripheral blood of NOG mice every 4 weeks after transplantation,and detect the differentiation of human immune cells by flow cytometry.2.Resveratrol was used as a positive control to test the effect of berberine on the in vitro expansion of human UCB-CD133~+HSCs.2.1 CD133~+HSCs were expanded in vitro using(0-50)μM resveratrol for 8 days,and cell proliferation activity was detected by MTS method.2.2 CD133~+HSCs were expanded in vitro with 10μM resveratrol for 0-14 days,and the proportion of CD34~+CD133~+cells before and after expansion was detected by flow cytometry.2.3 CD133~+HSCs were expanded in vitro using(0-20)μM berberine for 8 days,and the cell proliferation activity was detected by MTS method.2.4 CD133~+HSCs were expanded in vitro with(0-20)μM berberine for 8 days,and the proportion of CD34~+CD133~+cells before and after expansion was detected by flow cytometry.2.5 CD133~+HSCs were expanded in vitro with(0-10)μM berberine for 6-10 days,and the cell proliferation activity was measured by the MTS method.2.6 Use(1.25,2.5,5)μM berberine and(5,10)μM resveratrol to co-culture CD133~+HSCs for 8-10 days,use MTS method to detect cell proliferation activity.2.7 CD133~+HSCs were cultured with(5,10)μM berberine,10μM resveratrol,(5,10)μM berberine and 10μM resveratrol for 6-8 days.Use flow cytometry to detect the proportion of CD34~+CD133~+cells before and after culture.2.8 Use 5μM berberine,10μM resveratrol,5μM berberine and 10μM resveratrol were d to culture CD133~+HSCs for 6-8 days,Use Q-PCR to detect the relative expression of stem cell markers NANOG and SOX-2.Result1.Human UCB-CD133~+HSCs were transplanted into the bone marrow cavity of NOG mice pretreated with Busulfan to construct a humanized mouse model of the immune system and evaluate the construction effect;1.1 The percentage of CD133~+cells in one human UCB-MNCs was 40.7±0.11%,and the purity of CD133~+cells sorted by magnetic beads was 93.5±2.12%.1.2 One week after the transplantation(1 wpt),one mouse in the Busulfan pretreatment group showed behavioral changes such as hair wrinkles,humpback,gradual decrease in activity,and increased whole-body palpation intensity,and died at 4 wpt.Survival analysis showed that the survival rate of the control group(100%)was slightly higher than that of the Busulfan pretreatment group(83%),but there was no statistical difference(P>0.05).1.3 At 4 wpt,human CD45~+CD19~+cells and CD45~+CD3~-cells were detected from peripheral blood of NOG mice.Compared with the control group,this human B lymphocyte differentiation and the ratio of h-CD45~+to m-CD45~+in the peripheral blood of mice pretreated with Busulfan was significantly increased(P<0.01).1.4 At 16 wpt,human CD3~-,CD19~+,CD3~+,CD4~+and CD8~+cells were detected.The number of human lymphocyte differentiation and the ratio of human CD4~+to human CD8~+cells in the Busulfan pretreatment group were significantly higher than those in the control group(P<0.01).1.5 At 16 wpt,human CD3~-CD19~-CD14~-HLA-DR~+dendritic cells(DCs)and CD3~-CD19~-CD14~+monocytes were also detected.The number of differentiated DCs and monocytes in the Busulfan pretreatment group was 7 times and 3 times that of the control group,respectively.2.Resveratrol was used as a positive control to test the effect of berberine on the in vitro expansion of human UCB-CD133~+HSCs.2.1 Compared with the control group,10μM resveratrol can promote the proliferation activity of CD133~+HSCs(P<0.05),but the effect of maintaining the positive rate of CD133~+cells is significantly lower than that of CD34~+cells(P<0.0001).2.2 Compared with the 10μM resveratrol group,high concentration of berberine(10-20μM)can significantly increase the positive rate of CD133~+cells(P<0.0001),but it cannot increase the cell proliferation activity.2.3 Compared with the 10μM resveratrol group,low concentration berberine(5μM)is the best amplification concentration(P<0.01),5μM berberine and 10μM resveratrol are the best combined culture Concentration(P<0.01),both groups can increase the positive rate of CD133~+cells while promoting cell proliferation.8 days is the optimal number of culture days.2.4 Compared with the control group,5μM berberine significantly increased the relative expression of NANOG and SOX-2 genes when CD133~+HSCs were amplified in vitro for 8 days(P<0.0001).5μM berberine and 10μM resveratrol combination was the second most effective(P<0.05),and 10μM resveratrol could promote SOX-2 gene expression only after 8 days of culture(P<0.05).Conclusion1.Human UCB-CD133~+HSCs can be used as a good source for the establishment of humanized mouse models.Busulfan pretreatment combined with intramedullary injection can promote the differentiation of CD133~+HSCs in NOG mice.2.Berberine combined with SCF,TPO and Flt-3L cytokines can be used as an effective combination of reagents for in vitro amplification of human UCB-CD133~+HSCs,with the optimal concentration of 5μm and the maximum amplification period of 8 days.