In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

The immense regenerative power of hematopoietic tissue stems from the activation of the immature stem cells and the progenitor cells. After partial damage, hematopoiesis is reconstituted through a period of intense regeneration when blood cell production originates from erythro-myeloid progenitors in the virtual absence of stem cells. Since the damaged hematopoiesis can also be reconstituted from transplanted hematopoietic cells, we asked whether this also leads to the transient state when activated progenitors initially execute blood cell production. We first showed that the early reconstitution of hematopoiesis from transplanted cells gives rise to extended populations of developmentally advanced but altered progenitor cells, similar to those previously identified in the bone marrow regenerating from endogenous cells. We then identified the cells that give rise to these progenitors after transplantation as LSK CD48 – cells. In the submyeloablative irradiated host mice, the transplanted LSK CD48 – cells preferably colonized the spleen. Unlike the endogenous hematopoiesis reconstituting cells, the transplanted whole bone marrow cells and sorted LSK CD48 – cells had greater potential to differentiate to B-lymphopoiesis. Separate transplantation of the CD150 – and CD150 + subsets of LSK CD48 – cells suggested that CD150 – cells had a greater preference to B-lymphopoiesis than CD150 + cells. In the intensively regenerating hematopoiesis, the CD71/Sca-1 plot of immature murine hematopoietic cells revealed that the expanded populations of altered myeloid progenitors were highly variable in the different places of hematopoietic tissues. This high variability is likely caused by the heterogeneity of the hematopoiesis supporting stroma. Lastly, we demonstrate that during the period when active hematopoiesis resumes from transplanted cells, the hematopoietic tissues still remain highly permissive for further engraftment of transplanted cells, particularly the stem cells. Thus, these results provide a rationale for the transplantation of the hematopoietic stem cells in successive doses that could be used to boost the transplantation outcome.

Folia Biologica > Archive > 2021, Vol. 67 > Issue 4 > Latent Defect in Haematopoiesis of… Fol. Biol. 2021, 67, 135-135 https://doi.org/10.14712/fb2021067040135 Latent Defect in Haematopoiesis of UBC-GFP Mice Sheds Light on the Lymphoid Developmental Potential of Haematopoietic Stem Cells Emanuel Nečas, K. Faltusová, C-L. Chen Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Czech Republic Received November 2021 Accepted November 2021 Keywords stem cell, haematopoiesis, green fluorescent protein, UBC-GFP mouse, lymphopoiesis, T cell. References 4 live references Next article »

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.

Regeneration of severely damaged adult tissues is currently only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well established steady-state structure and function, easy accessibility, well established research methods, and the well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the need to expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. In the present study, we analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation and targeted the period of regeneration characterized by massive production of mature blood cells along with ongoing expansion of immature hematopoietic cells. We uncovered significantly expanded populations of developmentally advanced erythroid and myeloid progenitors with significantly altered immunophenotype. Their population expansion does not require erythropoietin stimulation but requires the SCF/c-Kit receptor signaling. Regenerating hematopoiesis significantly differs from the expanding hematopoiesis in the fetal liver but we find some similarities between the regenerating hematopoiesis and the early embryonic definitive hematopoiesis. These are in (1) the concomitant population expansion of myeloid progenitors and increasing production of myeloid blood cells (2) performing these tasks despite the severely reduced transplantation capacity of the hematopoietic tissues, and (3) the expression of CD16/32 in most progenitors. Our data thus provide a novel insight into tissue regeneration by suggesting that cells other than stem cells and multipotent progenitors can be of fundamental importance for the rapid recovery of tissue function.

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters. In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.

Tagging cells of experimental organisms with genetic markers is commonly used in biomedical research. Insertion of artificial gene constructs can be highly beneficial for research as long as this tagging is functionally neutral and does not alter the tissue function. The transgenic UBC-GFP mouse has been recently found to be questionable in this respect, due to a latent stem cell defect compromising its lymphopoiesis and significantly influencing the results of competitive transplantation assays. In this study, we show that the stem cell defect present in UBC-GFP mice negatively affects T-lymphopoiesis significantly more than B-lymphopoiesis. The production of granulocytes is not negatively affected. The defect in T-lymphopoiesis causes a low total number of white blood cells in the peripheral blood of UBC-GFP mice which, together with the lower lymphoid/myeloid ratio in nucleated blood cells, is the only abnormal phenotype in untreated UBCGFP mice to have been found to date. The defective lymphopoiesis in UBC-GFP mice can be repaired by transplantation of congenic wild-type bone marrow cells, which then compensate for the insufficient production of T cells. Interestingly, the wild-type branch of haematopoiesis in chimaeric UBC-GFP/wild-type mice was more active in lymphopoiesis, and particularly towards production of T cells, compared to the lymphopoiesis in normal wild-type donors.

Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation.

Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1(+) cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen.

Hematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analyzed the cell cycle and cell production rate in HSPCs in murine hematopoiesis. The labeling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled determination of the cell cycle flow rate into G2-phase, the duration of S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. Determination of cells with 2n DNA content labeled in preceding S-phase was then used to establish the cell flow rates in G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- myeloid progenitors self-renew and differentiate. Division of the Sca-1+ progenitors led to loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. Sca-1- cells arising from cell division entered a new round of the cell cycle, corresponding to symmetric self-renewing cell division. The novel data also enabled the estimation of the cell production rates in Sca-1+ and in three subtypes of Sca-1- HSPCs and revealed Sca-1 negative cells as the major amplification stage in the blood cell development.

We have previous studied the bone marrow response in submyeloablatively (6 Gy) irradiated mice. This allowed a detailed analysis of immature Lin-c-Kit+ (LK) cells only after approximately two weeks. These studies revealed significant expansion of Sca-1+ (S+) LK CD150+CD48+ cells with decreased c Kit expression level (Blood 2014 124:5112). Furthermore, they showed that not only LS+K cells responded to the injury but also Sca 1- (S-) late myeloid progenitors were activated and some of them were induced to re-expressed Sca 1 antigen. In present study, we focused on the response of LK cells occurring shortly after bone marrow damage by analyzing bone marrow 1-5 days after irradiation of mice with 2-4 Gy. The number of bone marrow cells significantly decreased after 2-4 Gy irradiation during one day. In LK cells, the LS-K/LS+K ratio decreased (below 2) due to Sca-1 induction. However, it recovered to normal level in a range of 5-7 after 3-5 days. We used CD150 and CD48 SLAM markers to distinguish between various subtypes of LSK cells, and CD34 and CD16/32 markers to characterize various types of LS-K myeloid progenitor cells. This revealed an early activation of megakaryocyte-erythroid and granulocyte macrophage myeloid progenitors. The activation was characterized by re-expression of Sca 1 in a part of these cells and by significantly decreased c-Kit expression level. During 3-5 days, c Kit recovered fully in 2 Gy- and partly in 4 Gy-irradiated mice. The transcription factor SCL/TAL1 with other co-factors is known to upregulate c-Kit gene and c-Kit expression. Therefore, we examined SCL/TAL1 mRNA level in LK cells in bone marrow of irradiated mice. Necas E, Szikszai Forgacova K, Faltusova K, et al: Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration. Blood 2014 124:5112 (abstract). Research was supported by the Grant Agency of the Czech Republic (GACR 17-01897S).

The hematopoiesis steadily generates a large number of blood cells by intensive cell proliferation. A hierarchy of hematopoietic stem and progenitor cells (HSPCs) has been well characterized in mouse. The aim of our study was to find out to which extent these distinct populations contributed to blood cell production. We estimated the production rates of various types of HSPCs according to the percentage of S phase cells, their number in bone marrow, and the S-phase duration. Flow cytometry was used for immunophenotyping of HSPCs according to Lin markers, c-Kit, Sca-1, CD48, CD150, CD34, CD16/32, CD71, and IL7R expression. The dual BrdU/EdU labelling technique was applied to determine the S-phase duration and the percentage of DNA-synthesizing (S-phase) HSPCs. The HSPC apoptotic rate was detected by Ghost Dye™ Red 780 staining. The S-phase fraction significantly differed in various types of HSPCs. The values ranged from ≈ 1 % to ≈ 90 % of cells in S-phase, and were a characterizing feature of particular types of HSPCs. The CD71 (transferrin receptor) expression level closely correlated with the proliferation rate in all types of HSPCs. The highest S-phase fraction was found in the Lin c Kit+Sca-1- progenitors committed to the megakaryocyte erythroid development, followed by the progenitors committed to the granulocyte macrophage development. The HSPCs lacking the Sca-1 antigen generated approximately 22 times more cells than the Sca 1 expressing ones. Research was supported by the Grant Agency of the Czech Republic (GACR 17-01897S).

We used transgenic mice expressing GFP under the human ubiquitin promoter (UBC-GFP mice; Schaefer et al, 2001) in experiments studying a spontaneous recovery of hematopoiesis damaged by submyeloablative irradiation. Results were strikingly different from corresponding experiments that used two congenic strains of mice CD45.2 and CD45.1. Analysis of this unexpected difference in results revealed that while UBC-GFP mice have normal hematopoiesis that also regenerates normally after damage, they are inferior when competing with transplanted bone marrow from wild-type mice (CD45.1 or CD45.2). Interestingly, UBC-GFP mice engrafted transplanted bone marrow of wild-type mice without conditioning, and their conditioning with a low sublethal dose of irradiation resulted in an inappropriately high level of engraftment of transplanted bone marrow of wild-type donors. Interestingly also, the wild-type mice origin hematopoietic and blood cells were consistently more represented in the peripheral blood, spleen and thymus as compared to the bone marrow. Transplanted bone marrow contributed both to myelopoiesis and lymphopoiesis but their contribution was significantly higher in the lymphoid cells. Chimeric hematopoiesis resulting from the transplantation of wild-type bone marrow to UBC-GFP recipients could be transplanted to secondary recipients confirming a defect in the lymphoid-biased hematopoietic stem cells in UBC-GFP mice. Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM: Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell Immunol. 2001 Dec 15;214(2):110-22. Research was supported by the Grant Agency od the Czech Republic (GACR 17-01897S).

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Cyclophosphamide (CY) alone or in combination with other drugs is widely used as a cytostatic and immunosuppressive agent in cancer patients. Together with total body irradiation or busulphan, CY is used for conditioning of bone marrow transplant recipients. It was shown that recovery of hematopoiesis from cyclophosphamide damage follows a unique progenitor regeneration pattern in mouse. In present paper, hematopoietic stem cells (HSCs) enriched in Lin-Sca1+ckit+ (LSK) and LSK CD48- CD150+ populations, their proliferation, apoptosis and competitive repopulation ability were determined in C57Bl/6 mice after single injection of CY in a submyeloablative dose 135 mg/kg. HSC recovery occurred in two oscillatory waves until day 20 after CY with two peaks vastly exceeding normal values on days 5 and 14. Significant increase in HSC proliferation activity preceded these peaks and a proliferative suppression occurred in between. After the first HSC peak on day 5, HSC numbers were normalized by their mobilization to peripheral blood. Return to normal values was not due to mobilization after the second peak on day 14 but HSC apoptosis rate increased up to 50%. In order to investigate the influence of p53-dependent apoptosis on this apoptosis upregulation in HSCs we used p53-/- mice injected by CY. Primary oscillation in HSC proliferation and numbers was identical to wild-type mice. Nevertheless, the secondary peaks were lower and increase of apoptosis was abrogated. We determined expression of mRNAs for SCF and SDF-1 in separated subsets of bone marrow stromal cells during regeneration after CY. While first wave of HSC regeneration was preceded by elevated expression of both cytokines and decline of SDF-1 mRNA was followed by mobilization, the later phases of regeneration seem to be regulated by distinct mechanisms. Cyclophosphamide even in moderate doses deregulates bone marrow microenvironment and causes prolonged disturbances in HSC seeding, proliferation, differentiation and apoptosis. Investigation of these processes can help us to understand better stem cell behavior.

Presence of the c-Kit tyrosine kinase receptor is a hallmark of the mouse hematopoietic stem cells (HSCs) and progenitors routinely used for their identification and separation. c-Kit is activated after binding of its ligand, the stem cell factor (SCF; c-Kit-ligand). c-Kit receptors with bound SCF form dimers that are rapidly internalized and degraded. This activates the c-Kit signaling pathways supporting cell survival, proliferation or quiescence and self-renewal. Although there is a consensus that c-Kit signaling is important for functioning of HSCs, published results are partly controversial. We have defined HSCs and progenitors as Lineage- Sca-1+c-Kit+ cells (LSK cells) and characterized them further by means of the CD150 and CD48 markers. We used anti-c-Kit antibody minus (FMO; Fluorescence Minus One) samples to distinguish between c-Kit+ and c-Kit- bone marrow cells and analyzed the distribution of c-Kit on the immature hematopoietic cells carrying different phenotypes. Further, we exposed bone marrow cells to a wide range of concentration of a recombinant mouse SCF in vitro and measured a change in c-Kit presence and distribution on these different cell types. Also, SCF was injected to mice in vivo and their bone marrow was similarly analyzed for a change in c-Kit expression. Bone marrow cells exposed to SCF concentrations that deeply down-regulated c-Kit receptors were transplanted to recipient mice, and their transplantation efficiency was compared to that of normal bone marrow. c-Kit was unevenly but characteristically distributed on different types of LSK CD150/CD48 cells, showing the highest and the most homogeneous density on cells with the LSK CD150+CD48- phenotype. Exposure of bone marrow cells to SCF in ranges of concentrations from 0.3-2000 ng/ml induced progressive down-regulation of c-Kit. However, the cells mostly remained c-Kit+(low). The response to SCF was the most prominent in a range of SCF concentrations between 1-100 ng/ml. Cells with the phenotype LSK CD150+CD48+were relative low-responders. In vivo administration of SCF to mice in doses exceeding 300 ng/mouse, either intraperitoneally or intravenously, had similar effect on c-Kit expression by bone marrow cells as their incubation with SCF in vitro. Next we investigated correlation of the intensity of c-Kit receptor expression on bone marrow cells with their repopulating capacity after transplantation. A significantly decreased c-Kit expression on transplanted cells, induced by exposure of the cells to SCF, did not decrease contribution of the cells to chimeric hematopoiesis in competitive transplantation assays. Formation of spleen colonies was also not affected in the CFU-S assay. Experiments which measured the effect of SCF administered to normal mice in vivo demonstrated an effect that lasted for less than 12 hours. c-Kit turnover on hematopoietic cells is thus rapid, and this fact may explain why down-regulation of c-Kit, on otherwise normal bone marrow cells, does not affect their capacity to be transplanted. In conclusion, c-Kit receptor density on hematopoietic cells does not appear to be a critical factor for the homing of transplanted hematopoietic stem and progenitor cells into the blood-forming tissues and their engraftment into specific niches. Also their performance in establishing productive hematopoiesis is not altered by the SCF-induced down-regulation of the c-Kit receptor density in time of their transplantation. Disclosures No relevant conflicts of interest to declare.

The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells.

Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma (NHL) associated with poor prognosis. Animal models of MCL are scarce. We established and characterized various in vivo models of metastatic human MCL by tail vein injection of either primary cells isolated from patients with MCL or established MCL cell lines (Jeko-1, Mino, Rec-1, Hbl-2, and Granta-519) into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. MCL infiltration was assessed with immunohistochemistry (tissues) and flow cytometry (peripheral blood). Engraftment of primary MCL cells was observed in 7 out of 12 patient samples. The pattern of engraftment of primary MCL cells varied from isolated involvement of the spleen to multiorgan infiltration. On the other hand, tumor engraftment was achieved in all five MCL cell lines used and lymphoma involvement of murine bone marrow, spleen, liver, and brain was observed. Overall survival of xenografted mice ranged from 22±1 to 54±3 days depending on the cell line used. Subsequently, we compared the gene expression profile (GEP) and phenotype of the engrafted MCL cells compared with the original in vitro growing cell lines (controls). We demonstrated that engrafted MCL cells displayed complex changes of GEP, protein expression, and sensitivity to cytotoxic agents when compared with controls. We further demonstrated that our MCL mouse models could be used to test the therapeutic activity of systemic chemotherapy, monoclonal antibodies, or angiogenesis inhibitors. The characterization of MCL murine models is likely to aid in improving our knowledge in the disease biology and to assist scientists in the preclinical and clinical development of novel agents in relapsed/refractory MCL patients.