Phenotypic profiling of CD34+ cells by advanced flow cytometry improves diagnosis of juvenile myelomonocytic leukemia.

Diagnostic criteria for juvenile myelomonocytic leukemia (JMML) are currently well defined, however in some patients diagnosis still remains a challenge. Flow cytometry is a well established tool for diagnosis and follow-up of hematological malignancies, nevertheless it is not routinely used for JMML diagnosis. Herewith, we characterized the CD34+ hematopoietic precursor cells collected from 31 children with JMML using a combination of standardized EuroFlow antibody panels to assess the ability to discriminate JMML cells from normal/reactive bone marrow cell as controls (n=29) or from cells of children with other hematological diseases mimicking JMML (n=9). CD34+ precursors in JMML showed markedly reduced B-cell and erythroid-committed precursors compared to controls, whereas monocytic and CD7+ lymphoid precursors were significantly expanded. Moreover, aberrant immunophenotypes were consistently present in CD34+ precursors in JMML, while they were virtually absent in controls. Multivariate logistic regression analysis showed that combined assessment of the number of CD34+CD7+ lymphoid precursors and CD34+ aberrant precursors or erythroid precursors had a great potential in discriminating JMMLs versus controls. Importantly our scoring model allowed highly efficient discrimination of truly JMML versus patients with JMML-like diseases. In conclusion, we show for the first time that CD34+ precursors from JMML patients display a unique immunophenotypic profile which might contribute to a fast and accurate diagnosis of JMML worldwide by applying an easy to standardize single eight-color antibody combination.

A novel flow-cytometric based method to assess post-HSCT donor chimerism exploiting RNA hybridization.

Analysis of donor-recipient chimerism after hematopoietic stem cell transplantation (HSCT) is of pivotal importance for patient's clinical management, especially in the context of mixed chimerism. Patients are routinely monitored for chimerism in sorted subsets of peripheral blood cells. However, measurement of chimerism in sorted immune cell subsets is technically challenging and time consuming. We here propose a novel, flow cytometry-based approach to detect donor cell chimerism in sex-mismatched HSCT. We exploit RNA PrimeFlow™ system, based on RNA hybridization, to detect mRNA from a lysine demethylase encoded by Y chromosome, KDM5D. This approach allows to distinguish male and female derived cells with around 1% sensitivity. The procedure can be coupled with multiparametric immunophenotyping to assess chimerism in specific immune cell subsets without the need for prior FACS-sorting. We apply this method to a cohort of HSCT patients (n = 10) and we show that it is consistent with standard PCR-based method. We also show that different T lymphocyte subsets display variable degrees of donor chimerism, especially in CD8+ T cell compartment where we observe an enrichment for recipient chimerism in central memory T cells. This method can be exploited to advance current knowledge on immune reconstitution focusing on specific subsets avoiding prior FACS-sorting.

Antibody Deficiency in Patients with Biallelic KARS1 Mutations.

Biallelic KARS1 mutations cause KARS-related diseases, a rare syndromic condition encompassing central and peripheral nervous system impairment, heart and liver disease, and deafness. KARS1 encodes the t-RNA synthase of lysine, an aminoacyl-tRNA synthetase, involved in different physiological mechanisms (such as angiogenesis, post-translational modifications, translation initiation, autophagy and mitochondrial function). Although patients with immune-hematological abnormalities have been individually described, results have not been collectively discussed and functional studies investigating how KARS1 mutations affect B cells have not been performed. Here, we describe one patient with severe developmental delay, sensoneurinal deafness, acute disseminated encephalomyelitis, hypogammaglobulinemia and recurrent infections. Pathogenic biallelic KARS1 variants (Phe291Val/ Pro499Leu) were associated with impaired B cell metabolism (decreased mitochondrial numbers and activity). All published cases of KARS-related diseases were identified. The corresponding authors and researchers involved in the diagnosis of inborn errors of immunity or genetic syndromes were contacted to obtain up-to-date clinical and immunological information. Seventeen patients with KARS-related diseases were identified. Recurrent/severe infections (9/17) and B cell abnormalities (either B cell lymphopenia [3/9], hypogammaglobulinemia [either IgG, IgA or IgM; 6/15] or impaired vaccine responses [4/7]) were frequently reported. Immunoglobulin replacement therapy was given in five patients. Full immunological assessment is warranted in these patients, who may require detailed investigation and specific supportive treatment.

Mutational analysis of ribosomal proteins in a cohort of pediatric patients with T-cell acute lymphoblastic leukemia reveals Q123R, a novel mutation in RPL10.

T-cell acute lymphoblastic leukemia (T-ALL) is a subtype of ALL involving the malignant expansion of T-cell progenitors. It is driven by a number of different possible genetic lesions, including mutations in genes encoding for ribosomal proteins (RPs). These are structural constituents of ribosomes, ubiquitous effectors of protein synthesis. Albeit the R98S mutation in RPL10, recurring with a higher frequency among RP mutations, has been extensively studied, less is known about the contribution of mutations occurring in other RPs. Alterations affecting translational machinery may not be well tolerated by cells, and there may be a selective pressure that determines the emergence of mutations with a compensatory effect. To explore this hypothesis, we sequenced the exomes of a cohort of 37 pediatric patients affected by T-ALL, and analyzed them to explore the co-occurrence of mutations in genes involved in ribosome biogenesis (including RPs) and translational control, and in known T-ALL driver genes. We found that some of the mutations in these sub-classes of genes tend to cluster together in different patients, indicating that their co-occurrence may confer some kind of advantage to leukemia cells. In addition, our sequencing highlighted the presence of a novel mutation in RPL10, namely the Q123R, which we found associated with a defect in protein synthesis. Our findings indicate that genetic alterations involving ribosome biogenesis and translational control should be carefully considered in the context of precision medicine in T-ALL.

Abnormal B-Cell Maturation and Increased Transitional B Cells in CBL Syndrome.

CBL syndrome is a Noonan-like RASopathy with heterogeneous clinical phenotype and predisposition to juvenile myelomonocytic leukemia (JMML). Here we describe two patients with identical germline CBL mutation and clinical and immune-hematological overlapping features with autoimmune lymphoproliferative syndrome (ALPS) and B-cell expansion with NF-κB and T-cell anergy (BENTA) syndrome. Increased immature/transitional B cells can be depicted in CBL syndrome, ALPS, and BENTA. Nonetheless, our patients here described showed peculiar B-cell phenotype due to increased immature/transitional CD34+ B cells. This feature differentiates CBL syndrome from BENTA, pointing toward an abnormal proliferation of B-cell early precursors.

Either IL-7 activation of JAK-STAT or BEZ inhibition of PI3K-AKT-mTOR pathways dominates the single-cell phosphosignature of ex vivo treated pediatric T-cell acute lymphoblastic leukemia cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from lymphoblasts of T-cell origin. While TALL accounts for only 15% of childhood and 25% of adult ALL, 30% of patients relapse with a poor outcome. Targeted therapy of resistant and high-risk pediatric T-ALL is therefore urgently needed, together with precision medicine tools allowing the testing of efficacy in patient samples. Furthermore, leukemic cell heterogeneity requires drug response assessment at the single-cell level. Here we used single-cell mass cytometry to study signal transduction pathways such as JAK-STAT, PI3K-AKT-mTOR and MEK-ERK in 16 diagnostic and five relapsed T-ALL primary samples, and investigated the in vitro response of cells to Interleukin-7 (IL-7) and the inhibitor BEZ-235. T-ALL cells showed upregulated activity of the PI3K-AKT-mTOR and MEK-ERK pathways and increased expression of proliferation and translation markers. We found that perturbation induced by the ex vivo administration of either IL-7 or BEZ-235 reveals a high degree of exclusivity with respect to the phospho-protein responsiveness to these agents. Notably, these response signatures were maintained from diagnosis to relapse in individual patients. In conclusion, we demonstrated the power of mass cytometry single-cell profiling of signal transduction pathways in T-ALL. Taking advantage of this advanced approach, we were able to identify distinct clusters with different responsiveness to IL-7 and BEZ-235 that can persist at relapse. Collectively our observations can contribute to a better understanding of the complex signaling network governing T-ALL behavior and its correlation with influence on the response to therapy.

Case Report: Hypomorphic Function and Somatic Reversion in DOCK8 Deficiency in One Patient With Two Novel Variants and Sclerosing Cholangitis.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient's disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the DOCK8 deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of DOCK8 may delay disease progression but do not necessarily prevent from severe complications.

Absent B cells, agammaglobulinemia, and hypertrophic cardiomyopathy in folliculin-interacting protein 1 deficiency.

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1-/- animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.

Single-cell profiling of pediatric T-cell acute lymphoblastic leukemia: Impact of PTEN exon 7 mutation on PI3K/Akt and JAK-STAT signaling pathways.

BACKGROUND: The PI3K/Akt/mTOR (PI3K) signaling pathway has a crucial role in T-cell acute lymphoblastic leukemias (T-ALLs). Although loss-of-function of phosphatase and tensin homolog (PTEN) is a common event in pediatric T-ALLs, the exact role of this tumor suppressor in T-ALL development has yet to be defined. METHODS: Here, we report an optimized cytometric method for accurate proteomic profiling of T-ALL leukemic blasts at single-cell level. We determined the expression of PI3K and JAK-STAT signaling components in both primary and immortalized T-ALL cells as well as in normal T cells. RESULTS: We observed that PTEN exon 7 mutated T-ALL cells retain a distinct PI3K activation; in particular, these cells show higher pAkt levels and a lower pS6 expression. Interestingly, we demonstrated for the first time that PTEN exon 7 mutated T-ALL are nonresponsive to IL7 in vitro as assessed by lack of pSTAT5 activation, although they do express IL7R. CONCLUSIONS: Phosphoflow analysis represents a fast, reliable, and accurate method to study the signaling profile of T-ALL. PTEN exon 7 mutated T-ALL cells are nonresponsive to IL7 in vitro suggesting that they may activate other mechanisms to support their viability and proliferation such as a higher constitutive PI3K/Akt signaling. Further investigations are necessary to elucidate the significance of this peculiar signaling behavior. Our observations should be taken into account in future studies aiming at molecular targeting of PI3K and/or JAK/STAT pathways for pharmacological intervention in T-ALL.

Metronomic combination of Vinorelbine and 5Fluorouracil is able to inhibit triple-negative breast cancer cells. Results from the proof-of-concept VICTOR-0 study.

Triple Negative Breast Cancer (TNBC) is an aggressive neoplasia with median Overall Survival (OS) less than two years. Despite the availability of new drugs, the chance of survival of these patients did not increase. The combination of low doses of drugs in a metronomic schedule showed efficacy in clinical trials, exhibiting an anti-proliferative and anti-tumour activity. In Victor-2 study we recently evaluated a new metronomic combination (mCHT) of Capecitabine (CAPE) and Vinorelbine (VNR) in breast cancer patients showing a disease control rate with a median Progression-Free Survival (PFS) of 4.7 months in 28 TNBC patients. Here in Victor-0 study, we examined the effect of mCHT vs standard (STD) schedule of administration of different combinations of 5-Fluorouracil (5FU), the active metabolite of CAPE, and VNR in TNBC cell lines MDA-MB-231 and BT-549. A significant anti-proliferative activity was observed in cells treated with metronomic vs STD administration of 5FU or VNR alone. Combination of the two drugs showed an additive inhibitor effect on cell growth in both cell lines. Moreover, after exposure of cells to 5FU and VNR under mCHT or conventional schedule of administration we also observed a downregulation of chemoresistance factor Bcl-2, changes in pro-apoptotic protein Bax and in cleaved effector caspase-3 and increased expression of LC3A/B autophagy protein. Our results therefore suggest that molecular mechanisms implicated in apoptosis and autophagy as well as the cross-talk between these two forms of cell death in MDA-MB-231 and BT-549 cells treated with 5FU and VNR is dose- and schedule-dependent and provide some insights about the roles of autophagy and senescence in 5FU/VNR-induced cell death.

SRC/ABL inhibition disrupts CRLF2-driven signaling to induce cell death in B-cell acute lymphoblastic leukemia.

Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the CRLF2 gene (hiCRLF2) have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.

Mesenchymal stromal cells from Shwachman-Diamond syndrome patients fail to recreate a bone marrow niche in vivo and exhibit impaired angiogenesis.

Shwachman-Diamond syndrome (SDS) is a rare multi-organ recessive disease mainly characterised by pancreatic insufficiency, skeletal defects, short stature and bone marrow failure (BMF). As in many other BMF syndromes, SDS patients are predisposed to develop a number of haematopoietic malignancies, particularly myelodysplastic syndrome and acute myeloid leukaemia. However, the mechanism of cancer predisposition in SDS patients is only partially understood. In light of the emerging role of mesenchymal stromal cells (MSCs) in the regulation of bone marrow homeostasis, we assessed the ability of MSCs derived from SDS patients (SDS-MSCs) to recreate a functional bone marrow niche, taking advantage of a murine heterotopic MSC transplant model. We show that the ability of semi-cartilaginous pellets (SCPs) derived from SDS-MSCs to generate complete heterotopic ossicles in vivo is severely impaired in comparison with HD-MSC-derived SCPs. Specifically, after in vitro angiogenic stimuli, SDS-MSCs showed a defective ability to form correct networks, capillary tubes and vessels and displayed a marked decrease in VEGFA expression. Altogether, these findings unveil a novel mechanism of SDS-mediated haematopoietic dysfunction based on hampered ability of SDS-MSCs to support angiogenesis. Overall, MSCs could represent a new appealing therapeutic target to treat dysfunctional haematopoiesis in paediatric SDS patients.

Simultaneous overexpression of human E5NT and ENTPD1 protects porcine endothelial cells against H2O2-induced oxidative stress and cytotoxicity in vitro.

Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5'-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig-to-non-human primates xenotransplantation models.

CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia.

Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL.We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers.Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated.Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib.In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.

The TGF-ß pathway is activated by 5-fluorouracil treatment in drug resistant colorectal carcinoma cells.

TGF-ß pathway is generally associated with the processes of metastasis, angiogenesis and EMT in cancer. Very little is known, however, about the role of TGF-ß in cancer drug resistance. In this work, we show a specific activation of the TGF-ß pathway in consequence of chemotherapeutic treatment in in vivo and in vitro models of colorectal carcinoma. 5-Fluorouracil (5FU) was able to stimulate the activation of SMAD3 and the transcription of specific genes such as ACVRL1, FN1 and TGFB1. On the other hand, the specific inhibition of TGF-ßRI was able to repress the 5FU-induced genes transcription and to restore the sensitivity of chemoresistant cells to the toxic action of the drug, by decreasing the expression of BCL2L1 and ID1 genes. The role of the TGF-ß molecule in the chemoresistant colon carcinoma cells' response to 5FU was further demonstrated by conditioned medium (CM) experiments: CM from 5FU-treated chemoresistant cells was able to protect chemosensitive cells against the toxic action of 5FU. In conclusion, these findings showed the pivotal role of TGF-ß pathway in colon cancer mechanisms of drug resistance suggesting new possible approaches in diagnosis and treatment of colon cancer patients.

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

LCK over-expression drives STAT5 oncogenic signaling in PAX5 translocated BCP-ALL patients.

The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2-3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2. More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.

PKH(high) cells within clonal human nephrospheres provide a purified adult renal stem cell population.

The existence and identification of adult renal stem cells is a controversial issue. In this study, renal stem cells were identified from cultures of clonal human nephrospheres. The cultured nephrospheres exhibited the activation of stem cell pathways and contained cells at different levels of maturation. In each nephrosphere the presence of 1.12-1.25 cells mirroring stem cell properties was calculated. The nephrosphere cells were able to generate three-dimensional tubular structures in 3D cultures and in vivo. In clonal human nephrospheres a PKH(high) phenotype was isolated using PKH26 epifluorescence, which can identify quiescent cells within the nephrospheres. The PKH(high) cells, capable of self-renewal and of generating a differentiated epithelial, endothelial and podocytic progeny, can also survive in vivo maintaining the undifferentiated status. The PKH(high) status, together with a CD133(+)/CD24(-) phenotype, identified a homogeneous cell population displaying in vitro self-renewal and multipotency capacity. The resident adult renal stem cell population isolated from nephrospheres can be used for the study of mechanisms that regulate self-renewal and differentiation in adult renal tissue as well as in renal pathological conditions.