Application Of An Ex Vivo Single HHSC Assay To Screening Of Small Molecule Compounds

Background:Hematopoietic stem cells (HSCs) are the stem cells which have been deeply investigated. HSCs can maintain stability of stem cell pool while differentiate into various cell populations due to their self-renewal and hierarchical differentiate potential. However, most studies and clinical trials are actually conducted with a mixture of differentiated cell subpopulations that include stem cells and more lineage-committed progenitors, owing to the high heterogeneity in blood cell populations and the rarity of HSCs. The identification of small molecule compounds that target HSCs is attractive in stem cell research due to the unique properties and structural diversity of these compounds. Among these small chemicals, SRI and UM171 offered the most promise for HSPC expansion, inducing even up to over 100-fold increase in HSPC number. Different combinations of cell surface markers enable the isolation of pure HSCs, flow cytometry now can provide an advanced multiparameter technique for separating and analyzing single cell. Researches showed that surface marker of HSCs including not only the classical marker CD34 and CD38, but also CD9^ CD45RA and specific marker of HSCs CD49f, this combination of surface marker could separate HSCs with HPCs. CFC and CAFC assays can be conducted to verify function of HSC, CFC could show differentiate ability of multipotent progenitors, furthermore, CAFC assay could reflect HSC that holds ex vivo long-term hematopoietic capacity.Objective:In this study, we used small molecule compounds SRI and UM171, which have been reported to have the ability of proliferating HSPC. We set up a platform for multiparameter sing-cell sorting. Furthermore, we employed ex vivo short-term and long-term functional assays to verify our screening platform, with the aim of applying this method for further screening of small molecule compounds.Methods:We used bulk culture to determine concentration of SRI and UM171. We constructed the best applied culture system for ex vivo single-cell culture, then sorted single DAPI-CD34+CD38-CD90+CD45RA-CD49f+ cell into wells, detected cell counts and phenotypes both by FCM and cell morphology assay after cultured for 2 weeks. Conducted CFC and CAFC assays to verify the stability of this system. Results:Determined compound concentration through CD34+cells bulk culture,SR1(1μMmol/L), UM171 (35nmol/L),SR1+UM171(750nmol/L+35nmol/L). After 14-day ex vivo single hHSC culture, flow cytometry resulted that treatment with SRI and UM171 showed an obvious increase when compared with the control group, both for wells formed colonies and absolute number of CD34+and CD34+CD49f+ cells(P<0.05). Cellilar morphology assays supported the flow cytometry results, almost all wells showed a balance differentiation to both lymphoid and myeloid cell lineages. CFC assays represented that treatment with small-molecule compounds significantly improved the colony-forming capacity of cultured cells. All kinds of colonies were found and there was no difference in the distribution or morphology of colonies between treatment and control group. CAFC assay showed compound-treated group enhanced the frequency of HSCs compared with control group (P<0.05).Conclusion:Treatment of sorted cells with SRI and UM171 in this ex vivo single hHSC assay showed that HSC could retain its self-renewal ability, at the same time, differentiated into different hematopoietic subpopulations. This approach could shorten the time-consuming and costly assay, primarily resolved heterogeneity of HSCs. Our studies thus provided a rapid and efficient single-HSC sorting and culture strategy for further applied to chemical screening.Background:Hematopoietic stem cells (HSCs) help keep the tissue homeostasis and give birth to all blood cells during both early development and the whole lifespan. The balance between HSCs self-renewal and multi-lineage differentiation plays an important role in HSCs physiological function. Recent researches showed an obvious evidence that this balance may partly be regulated by ROS, from both pluripotent embryonic and adult stem cell studies. ROS may mediate the cellular redox state, and by the way, realize synchrony with metabolism. Researches also show that, HSCs live a better life in a hypoxia environment, a low ROS level could help keep the long-repopulation ability of HSCs, then, help increase transplant survival rate. Clinical transplantation needs not only functional cells, but also enough HSCs, this is an urgent problem. The antioxidant defence system of human body is a very complex one, it can maintain ROS at a slight low level and ensure enough ROS for cell signaling, if this system cannot properly modulate the redox homeostasis, then external antioxidants are needed for the balance. Objective:Through primary screening of 85 antioxidants in small molecule compounds library, we got several compounds that do exact proliferation effect on HSCs. Then we verifed their effection on HSCs and explored their active antioxidant mechanism. Methods:For that all these small molecule compounds have not been reported in HSCs area, we conducted bulk CD34+cells ex vivo culture for primary screening, this method could help determin concentration of these compounds. After screened and a gradient concentration culture, absolute number and relative percentage of CD34+ cells and CD34+CD49ff cells were detected by flow cytometry, and a proper compound concentration was decided. Hematopoietic function experiment CFC (colony forming cell assay) then carried on to verify the effect of small molecule compounds on HSCs self-renewal and differentiation.Results:9 antioxidant small molecule compounds were achieved through primary screening, they were C2968 (5μM), B1753 (20μM)、B3358 (10μM)、C0168 (10μM)、 D3331 (5μMM)、B8071 (20μMM)、B8174 (10μM)、C0263 (20μMM) and C2943 (10μMM). Combined two important results, the concentration and proliferation effect, we finally chose five small molecule compounds for further grandient concentration culture:C2968 (5μMM)、B1753 (20μMM)、B3358 (10μMM)、 C0168 (10μM)、D3331 (5μMM). The final concentration is used for CFC assay. CFC showed that treatment with small molecule compounds C2968 (0.5μM、B1753 (1.5μM)、 B3358 (15μM)、 D3331 (1.5μM) could obviously increase colony forming ability of CD34+ cells(P<0.05), those compounds could retain multi-differentiate ability of progenitor cells especially multipotent progenitors.Conclusion:The antioxidant small molecule compound C2968 (0.5μM)、B1753(1.5μM)、B3358 (15μM)、 D3331 (1.5μM) showed a better ex vivo proliferation effect on HSCs, and balanced the self-renewal and multi-lineage differentiation. Through further research on its antioxidant mechanism, C2968 (0.5μM)、B1753 (1.5μM)、 B3358(15μM)、D3331 (1.5μM) can become a new tool for further understanding of the antioxidant defense mechanism of HSCs, and help not only fundamental research but also clinical transplantation of HSCs.