Treatment Of Ulcerative Colitis By Endometrial Regenerative Cells Through Regulation Of B Lymphocytes In Mice

Background: Ulcerative colitis(UC)is a chronic non-specific inflammatory disorder of colon with the persistent course.The pathophysiology is complex,and the imbalance of B cell function is thought to play an important role in the pathogenesis of ulcerative colitis.However,the effective treatment is still lacking.In recent years,stem cell therapy,as a promising tool,has been applied in the treatment of UC.Endometrial regenerative cells(ERCs)derived from menstrual blood,as a new source of stem cells,not only have the potential of self-renewal and mesenchymal stem cell(MSC)-like phenotypic characteristics,but also the immunomodulatory properities.Meanwhile,it possess the unique advantages,including the noninvasive obtaining procedure and the abundant source without ethical problems.Till now,whether ERCs could affect B cell function,and exert protective effect in colitis through the regulation of B cells remains to be elucidated.Objective: 1.To obtain ERCs from menstrual blood and establish the method system of separation,culture,passage,cryopreservation and resuscitation.2.To clarify the effect of ERCs on B cells in vitro.3.To investigate the immunoregulatory effect of ERCs on colitis mice.4.To observe the distribution of ERCs in colitis mice after injection.5.To explore whether ERCs exert protective effects through regulatory B cells.Methods:Part 1: Isolation of ERCs from menstrual blood.After informed consent was obtained,the menstrual blood was collected with Diva Cups at the 2nd to 3rd day of menstruation cycle from the healthy women donors.The mononuclear cell layer was separated by Ficoll-Paque density gradient centrifugation and subjected to passage culture in vitro.The immunophenotype of ERCs was analized by flow cytometry.Part 2: ERCs regulated B cell function in vitro.Mouse splenic CD19+ B lymphocytes were sorted by magnetic beads and cultured with LPS as stimulant in the presence or absence of ERCs.The proliferation of B cells was examined by 3H-thymidine incorporation.B cell viability was detected by trypan blue exclusion method,and the apoptosis of B cells was detected by flow cytometry analysis after staining with Annexin V and 7-AAD.B cell maturation/costimulatory markers surface expression were examined by flow cytometry analysis.The supernatant of the culture was collected and the contents of Ig M and Ig G in the supernatant were measured by ELISA.The percentage of IL-10 in the supernatant was detected by ELISA,and the percentage of IL-10+CD19+ B cells was detected by flow cytometry.Part 3: ERCs regulated the role of B cell function in colitis.Colitis was induced by admistering 3% dextran sodium sulfate(DSS)via free drinking water to Balb/c mice.6-8 weeks male Balb/c mice weighing 18-22 g were randomly divided into 3 groups(6-8 in each group):(1)normal control group: mice with free drinking water without DSS;(2)DSS-treated group: mice with free drinking 3% DSS solution for 7 days,followed by comsumption of normal water,injected of 200μl PBS at day 2,5 and 8 respectively through the tail vein;ERC-treated group: mice with free drinking 3% DSS solution for 7 days,followed by comsumption of normal water,injected of 200μl PBS containing 1 x 106 ERCs at day 2,5 and 8 respectively through the tail vein.Survival rates were recorded.The body weight was measured daily and the general condition,stool feature and the presence of gross stool blood were observed.DAI score was calculated to assess the disease activity.At the day10 after induction,mice were sacrificed.Colon length was measured and HE staining was performed to observe the pathological damage of colon.The protein level of pro-inflammatory factors in colon tissue homogenate was detected by ELISA.The proportion of immune cells in spleen,mesenteric lymph nodes and peritoneal lavage fluid was detected by flow cytometry analysis.Western blot and RT-PCR were used to detect the level of spleen IL-10.The distribution of PKH26 fluorescent labeled-ERCs after injection was examined by in vivo imaging detection system.The regulatory B cells were isolated and injected into colitis mice to explore whether ERCs played a protective role through these cells.Results:Part 1: Isolation of ERCs from menstrual blood.ERCs were obtained from the menstrual blood and separated by using Ficoll-Paque density gradient centrifugation.The primary adherent cells could be observed when the culture media was changed 3 days thereafter,and stretched into the spindle shape with culture gradually.It exhibited woven or spiral appearance at the confluence.After ERCs were passaged,the cell growth and proliferative rate accelerated,and the doubling time was about 24 hours.The biological and morphological characteristics became consistent at passage 3.Flow cytometry analysis showed that ERCs exhibited molecular phenotypes similar to MSCs with the expression CD90 and CD105 and the lack of hematopoietic stem cell markers CD34 and CD45.Meanwhile,the phenotype was still stable after passage,frozen and recovery.Part 2: ERCs regulated B cell function in vitro.ERCs inhibitted the proliferation of B lymphocytes induced by LPS in vitro without affecting B cell viability or inducing apoptosis.Meanwhile,ERCs suppressed the surface expression of mature/co-stimulatory markers of B cells and the secretion of antibodies in a dose-dependent manner.With the increase of ERCs/B cells ratio,the inhibitory effect was further enhanced.On the other hand,ERCs induced the production of immunosuppressive Bregs and IL-10 secretion.Part 3: ERCs regulated the function of B cells in colitis mice and attenuated colitis through Bregs.ERCs improved the survival rate of colitis mice,alleviating the body weight loss,delaying the deterioration of general condition and the occurance of diarrhea and the bloody stool later than mice in DSS-treated group.ERCs lightened the severity of colitis and promoted the recovery after changing with free drinking water.Colon length was longer in the ERC-treated group,and the gross pathological changes(intestinal rigidity,congestion,proximal colonic dilatation and distal colonic stenosis)were mild.HE staining conformed that ERCs could reduce the pathological changes of colon inflammation caused by DSS.Mucosal structure was relatively complete with less mucosal and submucosal inflammatory cell infiltration than that in DSS-treated group,with milder congestion and edema.At the same time,ERCs significantly reduced the levels of TNF-α、IL-1β and IL-6 in colitis mice and improved the level of IL-10.ERCs limitted the overwhelming proliferation of T cells in the spleen of colitis mice,reducing the proportion of CD3+ T cells,especially CD4+ T cells,while increasing the proportion of Tregs cell on the other hand.At the same time,ERCs inhibited splenic B cells to differentiate into plasma cells,reduced the deposition of Ig G antibodies in intestinal tissue of colitis mice,and decreased the content of Ig G in supernatant of intestinal homogenate.ERCs increased the proportion of IL-10+CD19+ Bregs from colitis mice and the proportion of CD1 dhi Bregs in spleen,peritoneal cavity and mesenteric lymph nodes.At the same time,the CD1dhiCD5+ Bregs in the spleen of the ERC-treated mice were significantly increased,and it was an important source of IL-10.Bregs could be induced by ERCs both in vivo and in vitro.ERC-pulsed Bregs effectively alleviated the symptoms of colitis and promoted intestinal recovery.ERCs was enriched in mouse colon,mesenteric lymph nodes,liver,kidney,spleen and lung.Above all,mesenteric lymph nodes and colon were the sites with most ERC enrichment,especially distal colon,consistent with the main characteristics of DSS induced colitis.Conclusions:1.Isolation and culture method of ERCs has been established and stable.ERCs shows spindle shape and MSC-like phenotype,which can be amplified in vitro.2.ERCs inhibit B cell proliferation,activation,antibody secretion,and induce Breg production.3.By using DSS-induced colitis model,we clarified that ERCs can reduce the symptoms of colitis,promote recovery,and reduce intestinal pathological damage and inflammatory response.4.ERCs are mainly distributed in the inflammatory reaction sites and lymphoid tissueof inflammatory drainage after injection into the colitis micel.5.ERCs regulate the balance of immune cells,inhibit the differentiation of B cells into plasma cells and antibody deposition in colon in DSS colitis model.On the other hand,ERCs induce Breg production in the spleen,peritoneal cavity and mesenteric lymph nodes.6.Adoptive tranfered ERC-pulsed Bregs effectively reduce colitis,indicating that ERCs may exert protective effects through Bregs.