All mice were housed in individually ventilated micro-isolator cages in the same room of certified SPF grade animal facility at IH. mice were cultured with M3434 medium for 14 days. Data shown are mean SEM of colony number (n?=?4).(TIFF) pone.0069913.s002.tiff (769K) GUID:?FD381C18-1C8A-48CD-8844-9BDF5E6E29B7 Figure S3: No alteration in differentiation abilities of HSCs from CL mice experiments. Furthermore, lymphoid differentiation of donor cells decreased while their myeloid differentiation increased under CL microenvironment although the overall level of donor hematopoietic repopulation was not significantly altered. Conclusions Our studies demonstrate that suppressing CPR expression enhances the repopulation efficiency of HSCs and a low CPR expression microenvironment favors the differentiation of myeloid over lymphoid lineage cells. Introduction The niche, and particularly its intracellular and extracellular redox metabolic microenvironment, is important for maintaining the self-renewal Ibuprofen Lysine (NeoProfen) and differentiation of hematopoietic stem cells (HSCs) [1], [2]. Under normal condition, HSCs that possess long-term reconstitution ability, namely long term-HSCs (LT-HSCs), reside in amicroenvironment with low PO2 Ibuprofen Lysine (NeoProfen) [3], [4], reportedly as low as 1% [5]. These HSCs express high level of Notch1, telomerase and Ibuprofen Lysine (NeoProfen) p21 [6]. About 70% HSCs are in the G0 phase, with low cell metabolic activity [7]. The low levels of metabolism, cell cycling and ROS are required for maintaining self-renewal capability for HSC and the alteration in the levels of metabolism or the damage to HSC reduces the self-renewal ability of HSC and may thus result in HSC exhaustion [8], [9]. NADPH-cytochrome P450 oxidoreductase (CPR) is an obligated electron donor for all microsomal cytochrome P450 (P450s or CYP) enzymes [10]. P450s are responsible for metabolizing many foreign compounds as well as endogenous substances [11]. CPR and P450 are also involved in the production of ROS. CPR and P450 are expressed in almost all tissues, including the bone marrow cells. In the absence of the functional Cpr gene, P450 are catalytically inactive. Germline deletion of the Cpr gene causes embryonic lethality in mice [12]. In humans, mutation leads to congenital steroidogenesis deficiency, which in turn may result in Antley-Bixler syndrome, characterized by skeletal malformation and reproductive defects [13]. We propose that CPR/P450 system may also be critical for hematopoiesis. In the current study, we used a genetically engineered mouse model with only 5%C24% CPR expression in various tissues (CL mice) [14] to examine the roles of CPR/P450 system in HSC hematopoiesis. Specifically, we compared the CL Rabbit Polyclonal to SLC25A6 mice with WT mice for their hematopoietic cell populations in the BM and PB, as well as the ability of HSCs for repopulation and differentiation using BM competitive transplantation and enriched HSC (LKS+) transplantation experiments. The impact of low CPR expression environment on hematopoiesis was examined by transplanting normal BM cells into CL recipients. The levels of ROS, cell cycle status, and apoptosis in the Ibuprofen Lysine (NeoProfen) BM were also compared between the CL and WT mice. Materials and Methods Mice C57BL/6J and B6.SJL were purchased from Vital River Laboratories (VRL, Beijing, China). The CL mice were generated and kindly provided by Dr. Xinxin Ding, Wadsworth Center, New York State Department of Health Albany, New York [14]. Briefly, the gene was disrupted by insertion of a gene in the intron 15 of the in CL mice, which led to a 74 to 95% decrease in CPR expression in all tissues examined, including olfactory mucosa, adrenal gland, brain, testis, ovary, lung, kidney, liver and heart. All mouse experiments were performed at the Institute of Hematology (IH), Tianjin, China. The mice used in the experiments have been backcrossed at least 10 times to the C57BL/6 background. If not specifically mentioned, sex matched WT and CL mice at 8C12 week-old were used in all the experiments. All mice were housed in individually ventilated micro-isolator cages in the same room of certified SPF grade animal facility at IH. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC), Institute of Hematology and Blood Disease Hospital, CAMS/PUMC. Antibodies for Flow Cytometry Antibodies against CD34 (Clone: RAM34), FLK2/FLT32 (Clone: A2F10.1),.