Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

Human lymphoid development in the absence of common γ-chain receptor signaling.

Despite the power of model systems to reveal basic immunologic mechanisms, critical differences exist between species that necessitate the direct study of human cells. Illustrating this point is the difference in phenotype between patients with SCID caused by mutations affecting the common γ-chain (γc) cytokine signaling pathway and mice with similar mutations. Although in both species, null mutations in either IL-2RG (which encodes γc), or its direct downstream signaling partner JAK3, result in T and NK cell deficiency, an associated B cell deficiency is seen in mice but not in humans with these genetic defects. In this study, we applied recent data that have revised our understanding of the earliest stages of lymphoid commitment in human bone marrow (BM) to determine the requirement for signaling through IL-2RG and JAK3 in normal development of human lymphoid progenitors. BM samples from SCID patients with IL-2RG (n = 3) or JAK3 deficiency (n = 2), which produce similar "T-NK-B+" clinical phenotypes, were compared with normal BM and umbilical cord blood as well as BM from children on enzyme treatment for adenosine deaminase-deficient SCID (n = 2). In both IL-2RG- and JAK3-SCID patients, the early stages of lymphoid commitment from hematopoietic stem cells were present with development of lymphoid-primed multipotent progenitors, common lymphoid progenitors and B cell progenitors, normal expression patterns of IL-7RA and TLSPR, and the DNA recombination genes DNTT and RAG1. Thus, in humans, signaling through the γc pathway is not required for prethymic lymphoid commitment or for DNA rearrangement.

Identification of perivascular mesenchymal stromal/stem cells by flow cytometry.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors of great promise for cell therapy. MSCs can mediate tissue regeneration, immunomodulation, and hematopoiesis support. Despite the unique properties of MSCs and their broad range of potential clinical applications, the very nature of these cells has been uncertain. Furthermore, MSCs are heterogeneous and only defined subpopulations of these are endowed with the particular abilities to sustain hematopoietic stem cells, regulate immune responses, or differentiate into mesodermal cell lineages. It is becoming evident that current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells. Here, we describe how flow cytometry has been used to reveal a perivascular origin of MSCs. As a result, the prospective purification of MSCs and specialized subsets thereof is now possible, and the clinical use of purified autologous MSCs is now within reach.

Novel pathways to erythropoiesis induced by dimerization of intracellular C-Mpl in human hematopoietic progenitors.

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl.

Dysregulated gene expression during hematopoietic differentiation from human embryonic stem cells.

The generation of hematopoietic cells from human embryonic stem cells (hESC) has raised the possibility of using hESC as an alternative donor source for transplantation. However, functional defects identified in hESC-derived cells limit their use for full lymphohematopoietic reconstitution. The purpose of the present study was to define and quantitate key functional and molecular differences between CD34(+) hematopoietic progenitor subsets derived from hESC and CD34(+) subsets from umbilical cord blood (UCB) representing definitive hematopoiesis. Two distinct sub-populations were generated following mesodermal differentiation from hESC, a CD34(bright) (hematoendothelial) and CD34(dim) (hematopoietic-restricted) subset. Limiting dilution analysis revealed profound defects in clonal proliferation relative to UCB particularly in B lymphoid conditions. Transcription factors normally expressed at specific commitment stages during B lymphoid development from UCB-CD34(+) cells were aberrantly expressed in hESC-derived CD34(+) cells. Moreover, strong negative regulators of lymphopoiesis such as the adaptor protein LNK and CCAAT/enhancer-binding protein-α (CEBPα), were exclusively expressed in hESC-CD34(+) subsets. Knockdown of LNK lead to an increase in hematopoietic progenitors generated from hESCs. The aberrant molecular profile seen in hESC-CD34(+) cells represents persistence of transcripts first expressed in undifferentiated hESC and/or CD326-CD56(+) mesoderm progenitors, and may contribute to the block in definitive hematopoiesis from hESC.

Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells.

Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelial-to-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326-CD56+ cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326-CD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326-CD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.

Lung alveolar integrity is compromised by telomere shortening in telomerase-null mice.

Shortened telomeres are a normal consequence of cell division. However, telomere shortening past a critical point results in cellular senescence and death. To determine the effect of telomere shortening on lung, four generations of B6.Cg-Terc(tm1Rdp) mice, null for the terc component of telomerase, the holoenzyme that maintains telomeres, were bred and analyzed. Generational inbreeding of terc-/- mice caused sequential shortening of telomeres. Lung histology from the generation with the shortest telomeres (terc-/- F4) showed alveolar wall thinning and increased alveolar size. Morphometric analysis confirmed a significant increase in mean linear intercept (MLI). terc-/- F4 lung showed normal elastin deposition but had significantly decreased collagen content. Both airway and alveolar epithelial type 1 cells (AEC1) appeared normal by immunohistochemistry, and the percentage of alveolar epithelial type 2 cells (AEC2) per total cell number was similar to wild type. However, because of a decrease in distal lung cellularity, the absolute number of AEC2 in terc-/- F4 lung was significantly reduced. In contrast to wild type, terc-/- F4 distal lung epithelium from normoxia-maintained mice exhibited DNA damage by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL) and 8-oxoguanine immunohistochemistry. Western blotting of freshly isolated AEC2 lysates for stress signaling kinases confirmed that the stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) stress response pathway is stimulated in telomerase-null AEC2 even under normoxic conditions. Expression of downstream apoptotic/stress markers, including caspase-3, caspase-6, Bax, and HSP-25, was also observed in telomerase-null, but not wild-type, AEC2. TUNEL analysis of freshly isolated normoxic AEC2 showed that DNA strand breaks, essentially absent in wild-type cells, increased with each successive terc-/- generation and correlated strongly with telomere length (R(2) = 0.9631). Thus lung alveolar integrity, particularly in the distal epithelial compartment, depends on proper telomere maintenance.

Identification of the critical extracellular matrix proteins that promote human embryonic stem cell assembly.

Human embryonic stem cells (hESC) exist as large colonies containing tightly adherent, undifferentiated cells. Disaggregation of hESC as single cells significantly affects their survival and differentiation, suggesting that adhesion mechanisms are critical for the assembly and maintenance of hESC colonies. The goal of these studies was to determine the key extracellular matrix (ECM) components that regulate assembly and growth of hESC. Our studies demonstrate that undifferentiated hESC express a specific subtype of laminin (laminin-511) and nidogen-1. The addition of a purified protein complex comprised of human laminin-511 and nidogen-1 to single-cell suspensions of hESC is sufficient to restore hESC assembly in the absence of murine embryonic fibroblasts or exogenous chemicals. The mechanism of hESC aggregation is through binding of the alpha6beta1 integrin receptor highly expressed in the membranes of undifferentiated hESC; aggregation can be inhibited by an antibody against alpha6 and almost completely blocked by an antibody against the beta1 subunit. Reassembly of defined numbers of purified hESC with the laminin-nidogen complex allows consistent production of uniform embryoid bodies (EBs) ("LN-EBs") that differentiate into endodermal, ectodermal, and mesodermal derivatives, and are highly efficient in generating hematoendothelial progenitors. These data reveal for the first time the crucial role of the ECM proteins laminin-511 and nidogen-1 in hESC assembly, and provide a novel practical tool to investigate hESC differentiation in a xenogen-free microenvironment.

Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element.

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Synergistic induction of DOC-2/DAB2 gene expression in transitional cell carcinoma in the presence of GATA6 and histone deacetylase inhibitor.

The down-regulation of DOC-2/DAB2 gene, which encodes a unique phosphoprotein modulating signal pathways elicited by exogenous stimuli, is often associated with several cancer types; however, the underlying mechanism is still unknown. Dramatically different expression levels of DOC-2/DAB2 mRNA and protein are observed among several human transitional cell carcinoma (TCC) cell lines, suggesting that transcriptional regulation may play a role in these cells. In this study, we have shown that the histone acetylation status associated with the 5' upstream regulatory sequence of DOC-2/DAB2 gene is one of the key determinants for its gene expression. In addition, GATA6 but not other GATA family members, such as GATA2 and GATA4, can specifically induce DOC-2/DAB2 promoter activity, although GATA transcription factors share a very similar DNA-binding sequence. We also show that increased histone acetylation and the presence of GATA6 have a synergistic effect on DOC-2/DAB2 promoter activity, which results in the elevation of DOC-2/DAB2 protein expression. Thus, we conclude that transcriptional regulation of DOC-2/DAB2 gene in human TCC is determined by histone acetylation and a specific transcription factor (i.e., GATA6), which underlie the reduced DOC-2/DAB2 protein expression in TCC cells.

Successful correction of the human beta-thalassemia major phenotype using a lentiviral vector.

beta-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human beta-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the beta-globin expression cassette. Thus far, mouse studies have shown correction of the beta-thalassemia intermedia phenotype and a partial, variable correction of beta-thalassemia major phenotype. We tested a lentiviral vector carrying the human beta-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human beta-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.

Characterization of a novel negative regulator (DOC-2/DAB2) of c-Src in normal prostatic epithelium and cancer.

DOC-2/DAB2 is a potent tumor suppressor in many cancer types including prostate cancer. In prostate cancer, expression of DOC-2/DAB2 can inhibit its growth. Our recent studies demonstrate that DOC-2/DAB2 can suppress both protein kinase C and peptide growth factor-elicited signal pathways via the Ras-mitogen-activated protein kinase pathway. In this study, we further showed that the proline-rich domain of DOC-2/DAB2 could also interact with proteins containing the Src homology 3 domain, such as Src and Fgr. The binding of c-Src to DOC-2/DAB2 was enhanced in cells treated with growth factor, and this interaction resulted in c-Src inactivation. The c-Src inactivation was evidenced by the decreased tyrosine 416 phosphorylation of c-Src and reduced downstream effector activation. It appears that DOC-2/DAB2 can bind to Src homology 3 domain of c-Src and maintain it in an inactive conformation. Thus, this study provides a new mechanism for modulating c-Src in prostatic epithelium and cancer.