Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.     

Reduced EBF expression underlies loss of B‐cell potential of hematopoietic progenitors with age

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age‐related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2–5‐fold, while myeloid potential remains unchanged. This age‐related decrease in B‐cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B‐lineage specifying factors, EBF and Pax5. Notably, retrovirus‐mediated expression of EBF complemented the age‐related loss of B‐cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B‐cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B‐cell fate, without altering their potential to generate myeloid cells.     

Osteoblast lineage cells expressing high levels of Runx2 enhance hematopoietic progenitor cell proliferation and function

Although osteoblasts (OB) play a key role in the hematopoietic stem cell (HSC) niche, little is known as to which specific OB lineage cells are critical for the enhancement of stem and progenitor cell function. Unlike hematopoietic cells, OB cell surface phenotypic definitions are not well developed. Therefore, to determine which OB lineage cells are most important for hematopoietic progenitor cell (HPC) function, we characterized OB differentiation by gene expression and OB function, and determined whether associations existed between OB and HPC properties. OB were harvested from murine calvariae, used immediately (fresh OB) or cultured for 1, 2, or 3 weeks prior to their co‐culture with Lin^?Sca1⁺c‐kit⁺ (LSK) cells for 1 week. OB gene expression, alkaline phosphatase activity, calcium deposition, hematopoietic cell number fold increase, CFU fold increase, and fold increase of Lin^?Sca1⁺ cells were determined. As expected, HPC properties were enhanced when LSK cells were cultured with OB compared to being cultured alone. Initial alkaline phosphatase and calcium deposition levels were significantly and inversely associated with an increase in the number of LSK progeny. Final calcium deposition levels and OB culture duration were inversely associated with all HPC parameters, while Runx2 levels were positively associated with all HPC properties. Since calcium deposition is associated with OB maturation and high levels of Runx2 are associated with less mature OB lineage cells, these results suggest that less mature OB better promote HPC proliferation and function than do more mature OB. J. Cell. Biochem. 111: 284–294, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of novel genes and networks governing hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome‐wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro. We report the discovery of novel genes important for the endothelial‐to‐hematopoietic transition and subsequently for HSPC specification. High‐throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.     

A novel ganglioside expressed by mouse hematopoietic cell lines.

Mouse progenitor T cell-derived cell lines were established by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW5147) to determine their characteristic cell-surface components. The hybridomas with the phenotype of Thy-1+, CD3-, CD4-, CD8- and expression of T cell receptor gene mRNA (BM216 and FL339) were selected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1a on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW5147). The structure of the ganglioside was determined to be: NeuAc alpha-Gal beta-Gal beta-Gal alpha-Gal beta-Glc beta-ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage. cells in hematopoietic organs committed to a specific     

LMO2与T细胞白血病

Lmo2基因是LMO(LIM-only)家族的成员之一。作为一个原癌基因,Lmo2的染色体异位t(11;14)(p13;q11)或t(7;11)(q35;p13)与T细胞急性淋巴细胞白血病密切相关。LMO2是细胞中介导转录因子复合物形成的重要接头分子。现对LMO2的分子结构及其在正常和白血病细胞中的调控作用机制的差异作重点介绍。在此基础上还讨论了LMO2成为逆转录病毒介导的基因治疗X染色体连锁的严重联合免疫缺陷综合征过程中成为病毒插入靶位点的可能原因。     

E2F4 modulates differentiation and gene expression in hematopoietic progenitor cells during commitment to the lymphoid lineage

The E2F4 protein is involved in gene repression and cell cycle exit, and also has poorly understood effects in differentiation. We analyzed the impact of E2F4 deficiency on early steps in mouse hematopoietic development, and found defects in early hematopoietic progenitor cells that were propagated through common lymphoid precursors to the B and T lineages. In contrast, the defects in erythromyeloid precursor cells were self-correcting over time. This suggests that E2F4 is important in early stages of commitment to the lymphoid lineage. The E2F4-deficient progenitor cells showed reduced expression of several key lymphoid-lineage genes, and overexpression of two erythromyeloid lineage genes. However, we did not detect effects on cell proliferation. These findings emphasize the significance of E2F4 in controlling gene expression and cell fate.     

EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Eph receptor (Eph)‐ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4–ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5⁺ cardiac progenitor cells and in α‐MHC⁺ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5‐GFP⁺ cardiac progenitor cells, but not in α‐MHC‐GFP⁺ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL‐RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full‐length EphB4 or intracellular domain‐truncated EphB4 in EphB4‐null ES cells was established to investigate the role of EphB4‐forward signaling in ES cells. Interestingly, while full‐length EphB4 was able to restore the cardiac lineage development in EphB4‐null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor‐kinase‐dependent EphB4‐forward signaling plays a crucial role in the development of cardiac progenitor cells. J. Cell. Biochem. 116: 467–475, 2015. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Active form of Notch imposes T cell fate in human progenitor cells

The crucial role of Notch signaling in cell fate decisions in hematopoietic lineage and T lymphocyte development has been well established in mice. Overexpression of the intracellular domain of Notch mediates signal transduction of the protein. By retroviral transduction of this constitutively active truncated intracellular domain in human CD34+ umbilical cord blood progenitor cells, we were able to show that, in coculture with the stromal MS-5 cell line, depending on the cytokines added, the differentiation toward CD19+ B lymphocytes was blocked, the differentiation toward CD14+ monocytes was inhibited, and the differentiation toward CD56+ NK cells was favored. The number of CD7+cyCD3+ cells, a phenotype similar to T/NK progenitor cells, was also markedly increased. In fetal thymus organ culture, transduced CD34+ progenitor cells from umbilical cord blood cells or from thymus consistently generated more TCR-gammadelta T cells, whereas the other T cell subpopulations were largely unaffected. Interestingly, when injected in vivo in SCID-nonobese diabetic mice, the transduced cells generated ectopically human CD4+CD8+ TCR-alphabeta cells in the bone marrow, cells that are normally only present in the thymus, and lacked B cell differentiation potential. Our results show unequivocally that, in human, Notch signaling inhibits the monocyte and B cell fate, promotes the T cell fate, and alters the normal T cell differentiation pathway compatible with a pretumoral state.     

Deep diving in the blood stem cell‐ome

Defining the functional distinctions between cells comprising the bone marrow has yielded fundamental insights into lineage ordering and drivers of blood cell production. A novel, highly granular and multi‐dimensional molecular characterization of functional subsets of hematopoietic stem‐ and progenitor cells recently published in Cell Stem Cell (Cabezas‐Wallscheid et al, 2014 ) will serve as a landmark and treasure trove for unanticipated insights into basic biology and the development of future targeted medicine.     

Pluripotent stem cell energy metabolism: an update

Recent studies link changes in energy metabolism with the fate of pluripotent stem cells (PSCs). Safe use of PSC derivatives in regenerative medicine requires an enhanced understanding and control of factors that optimize in vitro reprogramming and differentiation protocols. Relative shifts in metabolism from naïve through “primed” pluripotent states to lineage‐directed differentiation place variable demands on mitochondrial biogenesis and function for cell types with distinct energetic and biosynthetic requirements. In this context, mitochondrial respiration, network dynamics, TCA cycle function, and turnover all have the potential to influence reprogramming and differentiation outcomes. Shifts in cellular metabolism affect enzymes that control epigenetic configuration, which impacts chromatin reorganization and gene expression changes during reprogramming and differentiation. Induced PSCs (iPSCs) may have utility for modeling metabolic diseases caused by mutations in mitochondrial DNA, for which few disease models exist. Here, we explore key features of PSC energy metabolism research in mice and man and the impact this work is starting to have on our understanding of early development, disease modeling, and potential therapeutic applications.     

In vitro T lymphopoiesis: A model system for stem cell gene therapy for AIDS

Abstract: Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV infection. However, many important questions regarding the safety and efficacy of this approach remain unanswered and may be addressed in a non-human primate model. To facilitate evaluation of expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we have established a culture system that supports the differentiation of rhesus macaque and human CD34⁺ bone marrow derived cells into mature T cells. Thymic stromal monolayers were prepared from the adherent cell fraction of collagenase digested fetal or neonatal thymus. After 10–14 days, purified rhesus CD34⁺ bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3⁺CD4⁺CD8⁺, CD3⁺CD4⁺CD8^?, and CD3⁺CD4^?CD8⁺ cells. Following stimulation with mitogens, these T cells derived from CD34⁺ cells could be expanded over 1,000-fold and maintained in culture for up to 20 weeks. We next evaluated the ability of rhesus CD34⁺ cells transduced with a retroviral vector containing the marker gene neo to undergo in vitro T cell differentiation. CD34⁺ cells transduced in the presence of bone marrow stroma and then cultured on rhesus thymic stroma resulted in T cells containing the retroviral marker gene. These studies should facilitate both in vitro and in vivo studies of hematopoietic stem cell therapeutic strategies for AIDS.     

Neuropilin‐2 genomic elements drive cre recombinase expression in primitive blood,vascular and neuronal lineages

We have established a novel Cre mouse line, using genomic elements encompassing the Nrp2 locus, present within a bacterial artificial chromosome clone. By crossing this Cre driver line to R26R LacZ reporter mice, we have documented the temporal expression and lineage traced tissues in which Cre is expressed. Nrp2‐Cre drives expression in primitive blood cells arising from the yolk sac, venous and lymphatic endothelial cells, peripheral sensory ganglia, and the lung bud. This mouse line will provide a new tool to researchers wishing to study the development of various tissues and organs in which this Cre driver is expressed, as well as allow tissue‐specific knockout of genes of interest to study protein function. This work also presents the first evidence for expression of Nrp2 protein in a mesodermal progenitor with restricted hematopoietic potential, which will significantly advance the study of primitive erythropoiesis. genesis 53:709–717, 2015. © 2015 Wiley Periodicals, Inc.     

The contribution of the Tie2+ lineage to primitive and definitive hematopoietic cells

The regulatory elements of the Tie2/Tek promoter are commonly used in mouse models to direct transgene expression to endothelial cells. Tunica intima endothelial kinase 2 (Tie2) is also expressed in hematopoietic cells, although this has not been fully characterized. We determine the lineages of adult hematopoietic cells derived from Tie2‐expressing populations using Tie2‐Cre;Rosa26R‐EYFP mice. In Tie2‐Cre;Rosa26R‐EYFP mice, analysis of bone marrow cells showed Cre‐mediated recombination in 85% of the population. In adult bone marrow and spleen, we analyzed subclasses of early hematopoietic progenitors, T cells, monocytes, granulocytes, and B cells. We found that ～ 84% of each lineage was EYFP⁺, and nearly all cells that come from Tie2‐expressing lineages are CD45⁺, confirming widespread contribution to definitive hematopoietic cells. In addition, more than 82% of blood cells within the embryonic yolk sac were of Tie2⁺ origin. Our findings of high levels of Tie2‐Cre recombination in the hematopoietic lineage have implications for the use of the Tie2‐Cre mouse as a lineage‐restricted driver strain. genesis 48:563–567, 2010. © 2010 Wiley‐Liss, Inc.     

Protein profile of basal prostate epithelial progenitor cells—stage‐specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo

The long‐term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin^?Sca‐1⁺ CD49f⁺ Trop2^high‐phenotype) and human (Lin^? CD49f⁺ TROP2^high) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti‐human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single‐cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f⁺/TROP2^high phenotype of basal prostate progenitor cells and characterized by in vivo sandwich‐transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9⁺/CD24⁺/CD29⁺/CD44⁺/CD47⁺/CD49f⁺/CD104⁺/CD147⁺/CD326⁺/Trop2^high of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan‐1 and stage‐specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f⁺ TROP2⁺ basal prostate progenitor cells. Transplantation experiments suggest that CD49f⁺ TROP2^high SSEA‐4^high human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f⁺ TROP2^high or CD49f⁺ TROP2^high SSEA‐4^low cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA‐4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage.