CD45: a critical regulator in immune cells to predict severe and non-severe COVID-19 patients.

The ongoing outbreak of COVID-19 has been announced by the World Health Organization as a worldwide public health emergency. The aim of this study was to distinguish between severe and non-severe patients in early diagnosis. The results showed that the mortality of COVID-19 patients increased accompanied by age. Host factors CRP, IL-1ß, hs-CRP, IL-8, and IL-6 levels in severe pneumonia patients were higher than in non-severe patients. CD3, CD8, and CD45 counts were decreased in COVID-19 patients. The results of this study suggest that the K-values of CD45 might be useful in distinguishing between severe and non-severe cases. The cut-off value for CD45 was -94.33. The K-values for CD45 in non-severe case were above the cut-off values, indicating a 100% prediction success rate for severe and non-severe cases following SARS-CoV-2 infection. The results confirmed that immune system dysfunction is a potential cause of mortality following COVID-19 infection, particularly for the elderly. CD45 deficiency dysfunction the naïve and memory T lymphocytes which may affects the long-term effectiveness of COVID-19 vaccines. K-values of CD45 might be useful in distinguishing between severe and non-severe cases in the early infection. May be CD45 could increase the diagnostic sensitivity.

Lack of CD45 in FLT3-ITD mice results in a myeloproliferative phenotype, cortical porosity, and ectopic bone formation.

The receptor tyrosine kinase FLT3 is expressed in myeloid and lymphoid progenitor cells. Activating mutations in FLT3 occur in 25-30% of acute myeloid leukaemia (AML) patients. Most common are internal tandem duplications of sequence (ITD) leading to constitutive FLT3-ITD kinase activity with an altered signalling quality promoting leukaemic cell transformation. Here, we observed the attenuating role of the receptor-like protein tyrosine phosphatase (RPTP) CD45/Ptprc in FLT3 signalling in vivo. Low level expression of this abundant RPTP correlates with a poor prognosis of FLT3-ITD-positive AML patients. To get a further insight into the regulatory role of Ptprc in FLT3-ITD activity in vivo, Ptprc knock-out mice were bred with FLT3-ITD knock-in mice. Inactivation of the Ptprc gene in FLT3-ITD mice resulted in a drastically shortened life span and development of severe monocytosis, a block in B-cell development and anaemia. The myeloproliferative phenotype was associated with extramedullary haematopoiesis, splenohepatomegaly and severe alterations of organ structures. The phenotypic alterations were associated with increased transforming signalling of FLT3-ITD, including activation of its downstream target STAT5. These data reveal the capacity of Ptprc for the regulation of FLT3-ITD signalling activity in vivo. In addition, histopathology and computed tomography (CT) revealed an unexpected bone phenotype; the FLT3-ITD Ptprc-/- mice, but none of the controls, showed pronounced alterations in bone morphology and, in part, apparent features of osteoporosis. In the spleen, ectopic bone formation was observed. The observed bone phenotypes suggest a previously unappreciated capacity of FLT3-ITD (and presumably FLT3) to regulate bone development/remodelling, which is under negative control of CD45/Ptprc.

SR-BI (Scavenger Receptor Class B Type 1) Is Critical in Maintaining Normal T-Cell Development and Enhancing Thymic Regeneration.

Objective- Continuous T-cell production from thymus is essential in replenishing naïve T-cell pool and maintaining optimal T-cell functions. However, the underlying mechanisms regulating the T-cell development in thymus remains largely unknown. Approach and Results- We identified SR-BI (scavenger receptor class B type 1), an HDL (high-density lipoprotein) receptor, as a novel modulator in T-cell development. We found that SR-BI deficiency in mice led to reduced thymus size and decreased T-cell production, which was accompanied by narrowed peripheral naïve T-cell pool. Further investigation revealed that SR-BI deficiency impaired progenitor thymic homing, causing a dramatic reduction in the percentage of earliest thymic progenitors, but did not affect other downstream T-cell developmental steps inside the thymus. As a result of the impaired progenitor thymic homing, SR-BI-deficient mice displayed delayed thymic regeneration postirradiation. Using a variety of experimental approaches, we revealed that the impaired T-cell development in SR-BI-deficient mice was not caused by hematopoietic SR-BI deficiency or SR-BI deficiency-induced hypercholesterolemia, but mainly attributed to the SR-BI deficiency in adrenal glands, as adrenal-specific SR-BI-deficient mice exhibited similar defects in T-cell development and thymic regeneration with SR-BI-deficient mice. Conclusions- This study demonstrates that SR-BI deficiency impaired T-cell development and delayed thymic regeneration by affecting progenitor thymic homing in mice, elucidating a previously unrecognized link between SR-BI and adaptive immunity.

Loss of CD45 expression at relapse of acute myeloid leukemia.

A 49-year-old female was initially diagnosed with acute myeloid leukemia (AML) M4 with a CD45＋CD13＋CD33＋CD34-HLA-DR＋ immunophenotype. She underwent allogeneic bone marrow transplantation, but the disease recurred. The bone marrow was infiltrated with 87.0% blasts negative for myeloperoxidase (MPO) staining. Immunophenotyping by flow cytometry identified the presence of a CD45-negative blast population. These blasts exhibited a CD13＋CD33＋CD19-CD10-CD34-HLA-DR- immunophenotype. The lack of CD45 expression is often observed in B-cell acute lymphoblastic leukemia, whereas CD45-negative AML is extremely rare; only one older male with AML-M0 has been reported. In the present case, the CD45-negative blasts had an MPO-CD13＋CD33＋ phenotype, which is similar to AML-M0.

Dissecting and modeling the emergent murine TEC compartment during ontogeny.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45- ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII- CD80- → MHCIIlo CD80- → MHCIIhi CD80- → MHCIIhi CD80hi TECs, whereby MHCIIhi CD80- and MHCIIhi CD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhi CD80- cTECs directly generate mature MHCIIhi CD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

Single-cell molecular analysis defines therapy response and immunophenotype of stem cell subpopulations in CML.

Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA- fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a Lin-CD34+CD38-/lowCD45RA-cKIT-CD26+ population as a potential therapeutic target for improved therapy response.

CXCR4+ CD45- Cells are Niche Forming for Osteoclastogenesis via the SDF-1, CXCL7, and CX3CL1 Signaling Pathways in Bone Marrow.

Bone homeostasis comprises the balance between bone-forming osteoblasts and bone-resorbing osteoclasts (OCs), with an acceleration of osteoclastic bone resorption leading to osteoporosis. OCs can be generated from bone marrow cells (BMCs) under the tightly regulated local bone environment. However, it remained difficult to identify the critical cells responsible for providing an osteoclastogenesis niche. In this study, we used a fluorescence-activated cell sorting technique to determine the cell populations important for forming an appropriate microenvironment for osteoclastogenesis and to verify the associated interactions between osteoclast precursor cells and non-OCs. We isolated and removed a small cell population specific for osteoclastogenesis (CXCR4+ CD45- ) from mouse BMCs and cultured the remaining cells with receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage-colony stimulating factor. The resulting cultures showed significantly less large osteoclast formation. Quantitative RT-PCR analysis revealed that these CXCR4+ CD45- cells expressed low levels of RANK and RANKL, but high levels of critical chemokines including stromal cell derived factor 1 (SDF-1), chemokine (C-X-C motif) ligand 7 (CXCL7), and chemokine (C-X3-C motif) ligand 1 (CX3CL1). Furthermore, an SDF-1-specific antibody strongly suppressed OC formation in RAW264.7 cells and antibodies against SDF-1, CXCL7, and CX3CL1 suppressed OC formation in BMCs. These results suggest that isolated CXCR4+ CD45- cells support an appropriate microenvironment for osteoclastogenesis with a direct effect on the cells expressing SDF-1, CXCL7, and CX3CL1 receptors. The regulation of CXCR4+ CD45- cell function might therefore inform therapeutic strategies for diseases involving loss of bone homeostasis. Stem Cells 2016;34:2733-2743.

Aging induced loss of stemness with concomitant gain of myogenic properties of a pure population of CD34(+)/CD45(-) muscle derived stem cells.

Aging is accompanied by the functional decline of cells, tissues, and organs, as well as, a striking increase in susceptibility to a wide range of diseases. Within a tissue, both differentiated cells and adult stem cells are susceptible to intrinsic and extrinsic changes while aging. Muscle derived stem cells (MDSCs) are tissue specific stem cells which have been studied well for their multipotential nature. Although there are reports relating to diminished function and regenerative capacity of aged MDSCs as compared to their young counterparts, not much has been reported relating to the concomitant gain in unipotent nature of aged MDSCs. In this study, we report an inverse correlation between aging and expression of adult/mesenchymal stem cell markers and a direct correlation between aging and myogenecity in MDSCs. Aged MDSCs were able to generate a greater number of dystrophin positive myofibres, as compared to, the young MDSCs when transplanted in muscle of dystrophic mice. Our data, therefore, suggests that aging stress adds to the decline in stem cell characteristics with a concomitant increase in unipotency, in terms of, myogenecity of MDSCs. This study, hence, also opens the possibilities of using unipotent aged MDSCs as potential candidates for transplantation in patients with muscular dystrophies.

S-Nitrosoglutathione Reductase Deficiency Enhances the Proliferative Expansion of Adult Heart Progenitors and Myocytes Post Myocardial Infarction.

BACKGROUND: Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR(-/-)), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. METHODS AND RESULTS: GSNOR(-/-) and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR(-/-); n=3 WT) or MI (n=41 GSNOR(-/-); n=65 WT). Compared with WT, GSNOR(-/-) mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR(-/-) hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR(-/-) hearts demonstrated enhanced neovascularization (P<0.001), c-kit(+) CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit(+)/CD45(-) CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. CONCLUSIONS: Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.

Osteogenic differentiation of late-outgrowth CD45-negative endothelial progenitor cells.

BACKGROUND/AIMS: Late-outgrowth CD45-negative endothelial colony-forming cells are implicated to be circulating endothelial progenitor cells (EPCs), as they express endothelial cell markers and can directly form blood vessels. As these cells share characteristics of other progenitor cell phenotypes, late-outgrowth EPCs were assayed for both multilineage differentiation capability and for markers of pluripotency. METHODS: Clonal single-colony late-outgrowth EPCs were derived from human cord blood and assayed both for multilineage differentiation capability in vitro and for markers of pluripotency by qPCR. RESULTS: Under osteogenic growth conditions, these EPCs expressed the osteogenic markers RUNX2, COL1A1, ALPL, and osteocalcin and demonstrated calcium deposition and bone mineralization. Endothelial colony-forming cells expressed markers associated with induced pluripotent stem cells, including SOX2, POU5F1, c-MYC, and KLF4. CONCLUSIONS: Late-outgrowth EPCs express markers associated with pluripotency and can directly express an osteogenic phenotype under bone differentiation conditions in vitro.

CD90(+)CD45(-) intraperitoneal mesothelial-like cells inhibit T cell activation by production of arginase I.

In this study, we analyzed intraperitoneal cells recovered from human samples and found that CD90(+)CD45(-) cells exist as a minor population but vigorously grow in culture, showing the morphological features of mesothelial cells (MC). Interestingly, the MC highly expressed arginase I and markedly suppressed T cell proliferation with the reduction of CD3 ζ chain expression in T cells stimulated by coated anti-CD3 mAb. The addition of nor-NOHA (500 µM), or L-arginine (1 mM) mostly restored the inhibitory effect of MC on T cell proliferation as well as the reduced expression of CD3 ζ chain. The expression level of CD3 ζ chain in T cells in the peritoneal cavity was significantly down-regulated from circulating T cells. These results suggest that intraperitoneal free MC have immunomodulatory functions through the control of L-arginine level, and thus may play significant roles in the pathogenesis of various diseases in the peritoneal cavity.

Monocytes infiltrate the pancreas via the MCP-1/CCR2 pathway and differentiate into stellate cells.

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)(+)CD45(-) cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4). Because the vast majority of EGFP(+)CD45(-) cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP(+) PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C(high) monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP(+)F4/80(+)CCR2(+) monocytic cells and EGFP(+) PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP(+) bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP(+) PaSCs in injured mice. We propose that CCR2(+) monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.

Efficient xenoengraftment in severe immunodeficient NOD/Shi-scid IL2rγnull mice is attributed to a lack of CD11c+B220+CD122+ cells.

Xenograft animal models using immunodeficient mice have been widely applied in medical research on various human diseases. NOD/Shi-scid-IL2rγ(null) (NOG) mice are known to show an extremely high engraftment rate of xenotransplants compared with conventional immunodeficient mice. This high engraftment rate of xenotransplants in NOG mice was substantially suppressed by the transfer of spleen cells from NOD-scid mice that were devoid of NK cells. These results indicate that cell types other than splenic NK cells present in NOD-scid mice but not in NOG mice may be involved in this suppression. To identify the cell types responsible for this effect, we transferred subpopulations of spleen cells from NOD-scid mice into NOG mice and assessed the levels of human cell engraftment after human PBMC (hPBMC) transplantation. These experiments revealed that CD11c(+)B220(+) plasmacytoid dendritic cells (pDCs) from NOD-scid mice markedly inhibited engraftment of human cells. The CD11c(+)B220(+)CD122(+) cells further fractionated from the pDCs based on the expression of CD122, which is an NK cell marker strongly inhibited during hPBMC engraftment in NOG mice. Moreover, the CD122(+) cells in the pDC fraction were morphologically distinguishable from conventional CD122(+) NK cells and showed a higher rejection efficiency. The current results suggest that CD11c(+)B220(+)CD122(+) cells play an important role in xenograft rejection, and their absence in NOG mice may be critical in supporting the successful engraftment of xenotransplants.

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Isolation & characterization of Hoechst(low) CD45(negative) mouse lung mesenchymal stem cells.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Regulation of Src family kinases involved in T cell receptor signaling by protein-tyrosine phosphatase CD148.

CD148 is a receptor-like protein-tyrosine phosphatase known to inhibit transduction of mitogenic signals in non-hematopoietic cells. Similarly, in the hematopoietic lineage, CD148 inhibited signal transduction downstream of T cell receptor. However, it also augmented immunoreceptor signaling in B cells and macrophages via dephosphorylating C-terminal tyrosine of Src family kinases (SFK). Accordingly, endogenous CD148 compensated for the loss of the main SFK activator CD45 in murine B cells and macrophages but not in T cells. Hypothetical explanations for the difference between T cells and other leukocyte lineages include the inability of CD148 to dephosphorylate a specific set of SFKs involved in T cell activation or the lack of CD148 expression during critical stages of T cell development. Here we describe striking differences in CD148 expression between human and murine thymocyte subsets, the only unifying feature being the absence of CD148 during the positive selection when the major developmental block occurs under CD45 deficiency. Moreover, we demonstrate that similar to CD45, CD148 has both activating and inhibitory effects on the SFKs involved in TCR signaling. However, in the absence of CD45, activating effects prevail, resulting in functional complementation of CD45 deficiency in human T cell lines. Importantly, this is independent of the tyrosines in the CD148 C-terminal tail, contradicting the recently proposed phosphotyrosine displacement model as a mechanism of SFK activation by CD148. Collectively, our data suggest that differential effects of CD148 in T cells and other leukocyte subsets cannot be explained by the CD148 inability to activate T cell SFKs but rather by its dual inhibitory/activatory function and specific expression pattern.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Differential requirements for CD45 in NK-cell function reveal distinct roles for Syk-family kinases.

The protein tyrosine phosphatase CD45 is an important regulator of Src-family kinase activity. We found that in the absence of CD45, natural killer (NK) cells are defective in protecting the host from mouse cytomegalovirus infection. We show that although CD45 is necessary for all immunoreceptor tyrosine-based activation motif (ITAM)-specific NK-cell functions and processes such as degranulation, cytokine production, and expansion during viral infection, the impact of CD45 deficiency on ITAM signaling differs depending on the downstream function. CD45-deficient NK cells are normal in their response to inflammatory cytokines when administered ex vivo and in the context of viral infection. Syk and ζ chain-associated protein kinase 70 (Zap70) are thought to play redundant roles in transmitting ITAM signals in NK cells. We show that Syk, but not Zap70, controls the remaining CD45-independent, ITAM-specific NK-cell functions, demonstrating a functional difference between these 2 Syk-kinase family members in primary NK cells.

Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells.

Intestinal stem cells (ISCs) have been studied for more than three decades; however, their isolation has remained a challenge. We hypothesized that, just as for stem cells of other tissues, one or more membrane markers would allow positive selection of ISCs by antibody-based sorting. To explore this hypothesis, microarray data of putative ISC fractions generated by side population sorting and laser capture microdissection were subjected to bioinformatic analysis to identify common membrane antigens. The microarray comparison suggested CD24 as a candidate surface marker, and immunohistochemistry showed expression of CD24 in epithelial cells of crypt bases. Flow cytometry of jejunal epithelial preparations revealed a CD24(+) CD45(-) fraction comprising ∼1% of the cells. Analysis with epithelial cell adhesion molecule and CD31 confirmed that the cell preparations were epithelial and without endothelial contamination. Cycling cells identified by prior injection with 5-ethynyl-2'-deoxyuridine were found predominantly in the CD24(lo) subfraction. Transcript analysis by real-time RT-PCR showed this subfraction to be enriched in the ISC markers leucine-rich-repeat-containing G-protein-coupled receptor 5 (40-fold) and Bmi1 (5-fold), but also enriched in lysozyme (10-fold). Flow cytometry with anti-lysozyme antibodies demonstrated that Paneth cells comprise ∼30% of the CD24(lo) subfraction. Additional flow analyses with leucine-rich-repeat-containing G-protein-coupled receptor 5-enhanced green fluorescent protein (EGFP) epithelium demonstrated colocalization of EGFP(hi) and CD24(lo). In contrast, CD24 cells were negative for the quiescent ISC marker doublecortin and CaM kinase-like-1. Culture of CD24(lo) cells in Matrigel generated organoid structures, which included all four epithelial lineages, thus giving functional evidence for the presence of ISCs. We conclude that the CD24(lo) fraction of jejunal epithelium is highly enriched with cycling ISCs. This isolation method should be useful to many investigators in the field to advance both the basic understanding of ISC biology and the therapeutic applications of ISCs.