Correction: Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

[This corrects the article DOI: 10.1371/journal.pbio.1002526.].

Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.

NK cell's killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott-Aldrich syndrome protein, Wiskott-Aldrich syndrome protein-interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes.

Functional NK cell repertoires are maintained through IL-2Rα and Fas ligand.

Acquisition of a functional NK cell repertoire, known as education or licensing, is a complex process mediated through inhibitory receptors that recognize self. We found that NK cells containing self-killer Ig-like receptors for cognate HLA ligand in vivo were less susceptible to apoptosis. In vitro IL-15 withdrawal showed that uneducated NK cells upregulated Bim and Fas. Conversely, educated NK cells upregulated Fas ligand (FasL) under these conditions. Induction of cell death and Bim expression on uneducated cells correlated with increased IL-2Rα expression. Overexpression and knockdown studies showed that higher IL-2Rα limits NK cell survival in a novel manner that is independent from the role of IL-2 in activation-induced cell death. To study the role of FasL in induction of IL-2Rα(hi) NK cell death, a coculture assay with FasL-blocking Abs was used. IL-15 withdrawal led to FasL-dependent killing of IL-2Rα(hi) NK cells by more educated IL-2Rα(lo) NK cells. Finally, CMV reactivation induces a potent long-lasting population of licensed NK cells with enhanced survival. These findings show that education-dependent NK cell survival advantages and killing of uneducated NK cells result in the maintenance of a functional repertoire, which may be manipulated to exploit NK cells for cancer immunotherapy.

Notch signaling at later stages of NK cell development enhances KIR expression and functional maturation.

The Notch signaling pathway plays a substantial role in human NK cell development. However, the role of Notch on killer Ig-like receptor (KIR) upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood-derived NK cells or sorted peripheral blood NK cells were cultured with IL-15 for 7 d with inhibitory or activating Notch signals. Inhibition of Notch signaling significantly decreased KIR expression, whereas activation enhanced it. Overexpression of activated Notch on cord blood-derived NK cells resulted in a 2-fold increase in KIR expression, indicating that Notch signaling plays a direct, cell-intrinsic role in KIR regulation. Moreover, Notch-mediated KIR expression on NK cells is regulated through cis inhibition by delta-like ligand 1. Notch signaling also enhances CD16 upregulation that precedes KIR expression. Concomitant with the upregulation of KIR and CD16, Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even in posttransplant samples in which NK cell function is inherently defective. Given these attributes of Notch signaling, we propose that Notch agonists may enhance NK cell maturation and tumor killing in a posttransplant setting.

NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17).

The Fc receptor CD16 is present on essentially all CD56(dim) peripheral blood natural killer (NK) cells. Upon recognition of antibody-coated cells it delivers a potent signal to NK cells, which eliminate targets through direct killing and cytokine production. Here we investigated the regulation of CD16 surface expression after NK cell activation. Cytokine activation and target cell stimulation led to marked decreases in CD16 expression. Activation of CD56(dim) NK cells by cross-linking CD16 with antibodies resulted in a loss of CD16 and CD62L, which correlated with increased interferon-γ production. A disintegrin and metalloprotease-17 (ADAM17) is shown to be expressed by NK cells, and its selective inhibition abrogated CD16 and CD62L shedding, and led to enhanced interferon-γ production, especially when triggering was delivered through CD16. Fc-induced production of cytokines by NK cells exposed to rituximab-coated B cell targets was also enhanced by ADAM17 inhibition. This supports an important role for targeting ADAM17 to prevent CD16 shedding and improve the efficacy of therapeutic antibodies. Our findings demonstrate that over-activation of ADAM17 in NK cells may be detrimental to their effector functions by down-regulating surface expression of CD16 and CD62L.

Tim-3 is an inducible human natural killer cell receptor that enhances interferon gamma production in response to galectin-9.

NK-cell function is regulated by the integration of signals received from activating and inhibitory receptors. Here we show that a novel immune receptor, T-cell Ig and mucin-containing domain-3 (Tim-3), is expressed on resting human NK cells and is up-regulated on activation. The NK92 NK-cell line engineered to overexpress Tim-3 showed a marked increase in IFN-γ production in the presence of soluble rhGal-9 or Raji tumor cells engineered to express Gal-9. The Tim-3(+) population of low-dose IL-12/IL-18-activated primary NK cells significantly increased IFN-γ production in response to soluble rhGal-9, Gal-9 presented by cell lines, and primary acute myelogenous leukemia (AML) targets that endogenously express Gal-9. This effect is highly specific as Tim-3 Ab blockade significantly decreased IFN-γ production, and Tim-3 cross-linking induced ERK activation and degradation of IκBα. Exposure to Gal-9-expressing target cells had little effect on CD107a degranulation. Reconstituted NK cells obtained from patients after hematopoietic cell transplantation had diminished expression of Tim-3 compared with paired donors. This observation correlates with the known IFN-γ defect seen early posttransplantation. In conclusion, we show that Tim-3 functions as a human NK-cell coreceptor to enhance IFN-γ production, which has important implications for control of infectious disease and cancer.

Cutting edge: KIR antisense transcripts are processed into a 28-base PIWI-like RNA in human NK cells.

Killer Ig-like receptors (KIRs) are expressed in a variegated, clonally restricted fashion on NK cells and are important determinants of NK cell function. Although silencing of individual KIR genes is strongly correlated with the presence of CpG dinucleotide methylation within the promoter, the mechanism responsible for silencing has not been identified. Our results show that antisense transcripts mediate KIR transcriptional silencing through a novel PIWI-like 28-base small RNA. Although PIWI RNA-mediated silencing of transposable elements within germ cells have been described, this is the first report that identifies a PIWI-like RNA in an immune somatic cell lineage and identifies a mechanism that may be broadly used in orchestrating immune development.

TEM8 Tri-specific Killer Engager binds both tumor and tumor stroma to specifically engage natural killer cell anti-tumor activity.

BACKGROUND: The tumor microenvironment contains stromal cells, including endothelial cells and fibroblasts, that aid tumor growth and impair immune cell function. Many solid tumors remain difficult to cure because of tumor-promoting stromal cells, but current therapies targeting tumor stromal cells are constrained by modest efficacy and toxicities. TEM8 is a surface antigen selectively upregulated on tumor and tumor stromal cells, endothelial cells and fibroblasts that may be targeted with specific natural killer (NK) cell engagement. METHODS: A Tri-specific Killer Engager (TriKE) against TEM8-'cam1615TEM8'-was generated using a mammalian expression system. Its function on NK cells was assessed by evaluation of degranulation, inflammatory cytokine production, and killing against tumor and stroma cell lines in standard co-culture and spheroid assays. cam1615TEM8-mediated proliferation and STAT5 phosphorylation in NK cells was tested and compared with T cells by flow cytometry. NK cell proliferation, tumor infiltration, and tumor and tumor-endothelium killing by cam1615TEM8 and interleukin-15 (IL-15) were assessed in NOD scid gamma (NSG) mice. RESULTS: cam1615TEM8 selectively stimulates NK cell degranulation and inflammatory cytokine production against TEM8-expressing tumor and stromal cell lines. The increased activation translated to superior NK cell killing of TEM8-expressing tumor spheroids. cam1615TEM8 selectively stimulated NK cell but not T cell proliferation in vitro and enhanced NK cell proliferation, survival, and tumor infiltration in vivo. Finally, cam1615TEM8 stimulated NK cell killing of tumor and tumor endothelial cells in vivo. CONCLUSIONS: Our findings indicate that the cam1615TEM8 TriKE is a novel anti-tumor, anti-stroma, and anti-angiogenic cancer therapy for patients with solid tumors. This multifunctional molecule works by selectively targeting and activating NK cells by costimulation with IL-15, and then targeting that activity to TEM8+ tumor cells and TEM8+ tumor stroma.

The transcription factor c-Myc enhances KIR gene transcription through direct binding to an upstream distal promoter element.

The killer cell immunoglobulin-like receptor (KIR) repertoire of natural killer (NK) cells determines their ability to detect infected or transformed target cells. Although epigenetic mechanisms play a role in KIR gene expression, work in the mouse suggests that other regulatory elements may be involved at specific stages of NK-cell development. Here we report the effects of the transcription factor c-Myc on KIR expression. c-Myc directly binds to, and promotes transcription from, a distal element identified upstream of most KIR genes. Binding of endogenous c-Myc to the distal promoter element is significantly enhanced upon interleukin-15 (IL-15) stimulation in peripheral blood NK cells and correlates with an increase in KIR transcription. In addition, the overexpression of c-Myc during NK-cell development promotes transcription from the distal promoter element and contributes to the overall transcription of multiple KIR genes. Our data demonstrate the significance of the 5' promoter element upstream of the conventional KIR promoter region and support a model whereby IL-15 stimulates c-Myc binding at the distal KIR promoter during NK-cell development to promote KIR transcription. This finding provides a direct link between NK-cell activation signals and KIR expression required for acquisition of effector function during NK-cell education.

Low-density PD-1 expression on resting human natural killer cells is functional and upregulated after transplantation.

Expression of programmed cell death protein 1 (PD-1) on natural killer (NK) cells has been difficult to analyze on human NK cells. By testing commercial clones and novel anti-PD-1 reagents, we found expression of functional PD-1 on resting human NK cells in healthy individuals and reconstituting NK cells early after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Peripheral blood samples from healthy individuals and transplant recipients were stained for PD-1 expression using the commercial anti-PD-1 clone PD1.3.1.3, fluorescein isothiocyanate (FITC)-labeled pembrolizumab, or an FITC-labeled single-chain variable fragment (scFv) reagent made from pembrolizumab. These reagents identified low yet consistent basal PD-1 expression on resting NK cells, a finding verified by finding lower PD-1 transcripts in sorted NK cells compared with those in resting or activated T cells. An increase in PD-1 expression was identified on paired resting NK cells after allo-HSCT. Blockade of PD-1 on resting NK cells from healthy donors with pembrolizumab did not enhance NK function against programmed death-ligand 1 (PD-L1)-expressing tumor lines, but blocking with its scFv derivative resulted in a twofold increase in NK cell degranulation and up to a fourfold increase in cytokine production. In support of this mechanism, PD-L1 overexpression of K562 targets suppressed NK cell function. Interleukin-15 (IL-15) activity was potent and could not be further enhanced by PD-1 blockade. A similar increase in function was observed with scFv PD-1 blockade on resting blood NK cells after allo-HSCT. We identify the functional importance of the PD-1/PD-L1 axis on human NK cells in which blockade or activation to overcome inhibition will enhance NK cell-mediated antitumor control.

Potent Cytolytic Activity and Specific IL15 Delivery in a Second-Generation Trispecific Killer Engager.

Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.

Activated notch supports development of cytokine producing NK cells which are hyporesponsive and fail to acquire NK cell effector functions.

Natural Killer (NK) cells are powerful effectors of cytotoxicity against "stressed" cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34(+)/ICN(+) sorted cells resulted in a population of CD7(+) early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN(+) precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as interleukin (IL)-13, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN(+) precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 gamma, epsilon, and delta chains, RAG1/2, and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7(+) lymphoid precursor from UCB CD34(+) progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive, and poorly acquire NK cell receptors involved in self-tolerance and effector function.

Adaptive NK Cells Resist Regulatory T-cell Suppression Driven by IL37.

Natural killer (NK) cells are capable of fighting viral infections and cancer. However, these responses are inhibited by immune suppressor cells in the tumor microenvironment. Tumor progression promotes the recruitment and generation of intratumoral regulatory T cells (Treg), associated with a poor prognosis in cancer patients. Here, we show that canonical NK cells are highly susceptible to Treg-mediated suppression, in contrast to highly resistant CD57+ FcεRγ-NKG2C+ adaptive (CD56+CD3-) NK cells that expand in cytomegalovirus exposed individuals. Specifically, Tregs suppressed canonical but not adaptive NK-cell proliferation, IFNγ production, degranulation, and cytotoxicity. Treg-mediated suppression was associated with canonical NK-cell downregulation of TIM3, a receptor that activates NK-cell IFNγ production upon ligand engagement, and upregulation of the NK-cell inhibitory receptors PD-1 and the IL1 receptor family member, IL1R8 (SIGIRR or TIR8). Treg production of the IL1R8 ligand, IL37, contributed to the phenotypic changes and diminished function in Treg-suppressed canonical NK cells. Blocking PD-1, IL1R8, or IL37 abrogated Treg suppression of canonical NK cells while maintaining NK-cell TIM3 expression. Our data uncover new mechanisms of Treg-mediated suppression of canonical NK cells and identify that adaptive NK cells are inherently resistant to Treg suppression. Strategies to enhance the frequency of adaptive NK cells in the tumor microenvironment or to blunt Treg suppression of canonical NK cells will enhance the efficacy of NK-cell cancer immunotherapy. Cancer Immunol Res; 6(7); 766-75. ©2018 AACR.

161533 TriKE stimulates NK-cell function to overcome myeloid-derived suppressor cells in MDS.

Myelodysplastic syndrome (MDS) is a clonal heterogeneous stem cell disorder driven by multiple genetic and epigenetic alterations resulting in ineffective hematopoiesis. MDS has a high frequency of immune suppressors, including myeloid-derived suppressor cells (MDSCs), that collectively result in a poor immune response. MDSCs in MDS patients express CD155 that ligates the T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) and delivers an inhibitory signal to natural killer (NK) cells. To mediate a productive immune response against MDS, negative regulatory checkpoints, like TIGIT, expressed on MDS NK cells must be overcome. NK cells can be directed to lyse MDS cells by bispecific killer engagers (BiKEs) that ligate CD16 on NK cells and CD33 on MDS cells. However, such CD16 × CD33 (1633) BiKEs do not induce the proliferative response in MDS NK cells needed to sustain their function. Here, we show that the addition of an NK stimulatory cytokine, interleukin-15 (IL-15), into the BiKE platform leads to productive IL-15 signaling without TIGIT upregulation on NK cells from MDS patients. Lower TIGIT expression allowed NK cells to resist MDSC inhibition. When compared with 1633 BiKE, 161533 trispecific killer engager (TriKE)-treated NK cells demonstrated superior killing kinetics associated with increased STAT5 phosphorylation. Furthermore, 161533 TriKE-treated MDS NK cells had higher proliferation and enhanced NK-cell function than 1633 BiKE-treated cells without the IL-15 linker. Collectively, our data demonstrate novel characteristics of the 161533 TriKE that support its application as an immunotherapeutic agent for MDS patients.

Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices.

Transcriptional characterization of the Notch signaling pathway in rodent multipotent adult progenitor cells.

The Notch signaling pathway is a multifunctional, evolutionarily conserved pathway, which plays an important role in development as well as stem cell biology. Multipotent adult progenitor cells (MAPCs) represent a unique stem cell population, which is capable of differentiating into cell types of the ectodermal, mesodermal and endodermal lineages in vitro, and contribute to most somatic cell types in vivo. Our aim was to characterize the gene expression of Notch signaling elements in rodent MAPCs. We show that transcripts for Notch-receptors, ligands, regulatory molecules of the pathway and the Hairy/Enhancer of Split-1 (HES-1) target gene are present in mouse and rat low-Oct4 MAPCs. We found that mouse Notch3 and rat Notch1 transcripts increased when cells were cultured at high density for 48 to 96 hours. HES-1 and HES-related transcription factor-1 (HERP-1), transcriptional targets of Notch-signaling, were both elicited by immobilized Delta1 ligand. In addition, mRNA for Notch1 and Notch3 was also induced by Notch-signaling, suggesting the presence of regulatory feedback loops. Slight differences between mouse and rat derived MAPCs suggest that the exact function, transcriptional regulation and the fine-tuning of the signal may be species specific. Taken together, we characterized the gene expression profile of the Notch pathway in rodent low-Oct4-MAPCs, and showed that the pathway is functional and can be modulated. Our results provide an additional tool and a further basis for a better understanding of stem cell biology.

Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells.

Cancer immunotherapies have gained significant momentum over the past decade, particularly with the advent of checkpoint inhibitors and CAR T-cells. While the latter personalized targeted immunotherapy has revolutionized the field, a need for off-the-shelf therapies remains. The ability of NK cells to quickly lyse antibody-coated tumors and potently secrete cytokines without prior priming has made NK cells ideal candidates for antigen-specific immunotherapy. NK cells have been targeted to tumors through two main strategies: mono-specific antibodies and bi/tri-specific antibodies. Mono-specific antibodies drive NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Bi/tri-specific antibodies drive re-directed lysis of tumor cells through binding of a tumor antigen and direct binding and crosslinking of the CD16 receptor on NK cells, thus bypassing the need for binding of the Fc portion of mono-specific antibodies. This chapter focuses on the generation of bi- and tri-specific killer engagers (BiKEs and TriKEs) meant to target NK cells to tumors. BiKEs and TriKEs are smaller molecules composed of 2-3 variable portions of antibodies with different specificities, and represent a novel and more versatile strategy compared to traditional bi- and tri-specific antibody platforms.

Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain.

OBJECTIVE: Recent studies have shown that cells from bone marrow (BM), muscle, and brain may have greater plasticity than previously known. We have identified multipotent adult progenitor cells (MAPC) in postnatal human and rodent BM that copurify with mesenchymal stem cells (MSC). BM MAPC proliferate without senescence and differentiate into mesodermal, neuroectodermal, and endodermal cell types. We hypothesized that cells with characteristics similar to BM MAPC can be selected and cultured from tissues other than BM. MATERIALS AND METHODS: BM, whole brain, and whole muscle tissue was obtained from mice. Cells were plated on Dulbecco modified Eagle medium supplemented with 2% fetal calf serum and 10 ng/mL epidermal growth factor (EGF), 10 ng/mL platelet-derived growth factor (PDGF-BB), and 1000 units/mL leukemia inhibitory factor (LIF) for more than 6 months. Cells were maintained between 0.5 and 1.5 x 10(3) cells/cm(2). At variable time points, we tested cell phenotype by FACS and evaluated their differentiation into endothelial cells, neuroectodermal cells, and endodermal cells in vitro. We also compared the expressed gene profile in BM, muscle, and brain MAPC by Affimetrix gene array analysis. RESULTS: Cells could be cultured from BM, muscle, and brain that proliferated for more than 70 population doublings (PDs) and were negative for CD44, CD45, major histocompatibility complex class I and II, and c-kit. Cells from the three tissues differentiated to cells with morphologic and phenotypic characteristics of endothelium, neurons, glia, and hepatocytes. The expressed gene profile of cells derived from the three tissues was identical (r(2) > 0.975). CONCLUSIONS: This study shows that cells with MAPC characteristics can be isolated not only from BM, but also from brain and muscle tissue. Whether MAPC originally derived from BM are circulating or all organs contain stem cells with MAPC characteristics currently is being studied. Presence of MAPC in multiple tissues may help explain the "plasticity" found in multiple adult tissues.