Induced overexpression of OCT4A in human embryonic stem cells increases cloning efficiency.

Our knowledge of the molecular mechanisms underlying human embryonic stem cell (hESC) self-renewal and differentiation is incomplete. The level of octamer-binding transcription factor 4 (Oct4), a critical regulator of pluripotency, is precisely controlled in mouse embryonic stem cells. However, studies of human OCT4 are often confounded by the presence of three isoforms and six expressed pseudogenes, which has complicated the interpretation of results. Using an inducible lentiviral overexpression and knockdown system to manipulate OCT4A above or below physiological levels, we specifically examine the functional role of the OCT4A isoform in hESC. (We also designed and generated a comparable series of vectors, which were not functional, for the overexpression and knockdown of OCT4B.) We show that specific knockdown of OCT4A results in hESC differentiation, as indicated by morphology changes, cell surface antigen expression, and upregulation of ectodermal genes. In contrast, inducible overexpression of OCT4A in hESC leads to a transient instability of the hESC phenotype, as indicated by changes in morphology, cell surface antigen expression, and transcriptional profile, that returns to baseline within 5 days. Interestingly, sustained expression of OCT4A past 5 days enhances hESC cloning efficiency, suggesting that higher levels of OCT4A can support self-renewal. Overall, our results indicate that high levels of OCT4A increase hESC cloning efficiency and do not induce differentiation (whereas OCT4B expression cannot be induced in hESC), highlighting the importance of isoform-specific studies in a stable and inducible expression system for human OCT4. Additionally, we demonstrate the utility of an efficient method for conditional gene expression in hESC.

Genetic modification of airway progenitors after lentiviral gene delivery to the amniotic fluid of murine fetuses.

Lentiviral vectors with the firefly luciferase or enhanced green fluorescent protein (EGFP) transgenes were delivered to the amniotic fluid of murine fetuses at Embryonic Day (E) 14.5 or E16.5. Whole-body imaging of luciferase recipients after birth demonstrated transgene expression in the peritoneal and thoracic regions. Organ imaging showed luciferase expression in lung, skin, stomach, and/or intestine. Histological immunofluorescence analysis of EGFP recipients demonstrated that small clusters (≤ three cells) of EGFP-positive epithelial cells were present in the large and small airways of recipients at up to 7 months (n = 11). There was no difference in the frequency of transgene expression in mice injected at E14.5 or E16.5 in respiratory or nonrespiratory organs. Analysis of the bronchoalveolar duct junctions on tissue sections of recipient mice identified multiple EGFP-positive epithelial cells. Cells coexpressing EGFP, Clara cell 10-kd protein, and surfactant protein C (SPC) were also found in lungs, consistent with the transduction of bronchoalveolar stem cells. Next, naphthalene lung injury in both luciferase and EGFP recipients was performed to determine whether transduced cells could contribute to tissue repair. In luciferase recipients, the whole-body luciferase signal increased 2- to 20-fold at 2 weeks after naphthalene treatment. Remarkably, immunohistological analysis of the lungs of EGFP recipients after lung injury repair demonstrated repopulation of airways with long stretches of EGFP-positive epithelial cells (n = 4). Collectively, these data demonstrate that lentiviral gene delivery to the amniotic fluid of murine fetuses genetically modifies long-lived epithelial progenitors capable of contributing to lung injury repair.

Recovery of multipotent progenitors from the peripheral blood of patients requiring extracorporeal membrane oxygenation support.

RATIONALE: Studies have demonstrated that bone marrow-derived cells can be recruited to injured lungs through an unknown mechanism. We hypothesize that marrow progenitors are mobilized into the circulation of patients with cardiac and/or respiratory failure, and may then traffic to and incorporate into the sites of tissue injury. OBJECTIVES: To determine whether progenitor populations are increased in the blood of patients with severe acute cardiorespiratory failure placed on extracorporeal membrane oxygenation (ECMO). METHODS: Mononuclear cells from ECMO, umbilical cord, and control blood samples were evaluated in colony-forming assays for hematopoietic, mesenchymal, and epithelial cells. Progenitors were identified by proliferative and differentiative capacities, and confirmed by the expression of lineage-specific markers. MEASUREMENTS AND MAIN RESULTS: Significantly higher levels of hematopoietic progenitors were observed in ECMO (n = 41) samples than neonatal intensive care unit (n = 16) or pediatric intensive care unit controls (n = 14). Hematopoietic progenitor mobilization increased with time on ECMO support. Mesenchymal progenitors (MSC) were recovered from 18/58 ECMO samples with rapid sample processing (< 4 h) critical to their recovery. MSC were not recovered from normal controls. ECMO-derived MSC had osteogenic, chondrogenic, and adipogenic differentiation potential. The recovery of MSC did not influence survival outcome (61%). Epithelial progenitors were observed in eight ECMO samples but not in control samples. Their presence was associated with a lower survival trend (38%). CONCLUSIONS: Hematopoietic, mesenchymal, and epithelial progenitors were mobilized into the circulation of patients on ECMO. This may reflect a response to severe cardiopulmonary injury, blood-foreign surface interactions with the ECMO circuit, and/or hemodilution.

Molecular characterization of the human NANOG protein.

NANOG is a key transcriptional regulator of pluripotent stem cell (PSC) self-renewal. NANOG occupies promoters that are active and others that are repressed during self-renewal; however, the mechanisms by which NANOG regulates transcriptional repression and activation are unknown. We hypothesized that individual protein domains of NANOG control its interactions with both the promoters and its coregulators. We performed a detailed characterization of the functional domains in the human (h) NANOG protein, using a panel of deletion-mutant and point-mutant constructs. We determined that six amino acids in the homeodomain ((136)YKQVKT(141)) are sufficient for the nuclear localization of hNANOG. We also determined that the tryptophan-rich region (W) of hNANOG contains a CRM1-independent signal for nuclear export, suggesting a possible cellular shuttling behavior that has not been reported for hNANOG. We also show that at least four tryptophans are required for nuclear export. We also determined that similar to murine (m) NANOG, the W region of hNANOG contains a homodimerization domain. Finally, in vitro transactivation analyses identified distinct regions that enhance or diminish activity at gene promoters that are active during self-renewal. Specifically, the N-terminal region interferes with transcription and removal of this region that produced a "super-active" hNANOG with enhanced transcriptional activity. We also confirmed that the transcriptional activator in hNANOG is contained in the C-terminal region, similar to murine NANOG. In summary, this study has characterized the structure and function of hNANOG protein leading to an increased understanding of the mechanism by which hNANOG regulates both transcriptional activation and repression during PSC self-renewal.

Protein kinase CKIIalpha interacts with the Bcr moiety of Bcr/Abl and mediates proliferation of Bcr/Abl-expressing cells.

The Bcr protein was originally identified because of its fusion to Abl as a consequence of the Philadelphia chromosome translocation found in chronic myelogenous and acute lymphoblastic leukemias. The Bcr moiety is essential for the transforming activity of the Bcr/Abl oncogene. In search of physiologically relevant Bcr and Bcr/Abl-interacting proteins, we performed an interaction screen in yeast using the entire Bcr protein as bait. We here report that the alpha catalytic subunit of protein kinase CKII strongly and specifically forms a complex with Bcr in yeast in mouse lysates. The region in Bcr responsible for CKIIalpha binding was localized to residues 242-413. CKIIalpha was previously shown to be involved in leukemogenesis and tumorigenesis using different experimental approaches including mouse models. Inhibition of Bcr/Abl P190 in lymphoma cells from Bcr/Abl transgenic mice using imatinib reduced CKIIalpha activity. A highly selective inhibitor of CKIIalpha, 4,5,6,7-tetrabromo-2-benzotriazole, inhibited the growth of murine lymphoid cells with induced P210 Bcr/Abl expression and of P190 lymphoma cells. Our results demonstrate that CKIIalpha plays an important role in the proliferation of Bcr/Abl expressing cells, and suggests that inhibitors of CKIIalpha may have therapeutic potential in the treatment of Bcr/Abl-positive leukemia patients.

Scambio, a novel guanine nucleotide exchange factor for Rho.

BACKGROUND: Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. RESULTS: Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. CONCLUSION: Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

Interaction of the small GTPase Rac3 with NRBP, a protein with a kinase-homology domain.

The family of Rac small GTPases including Rac1, Rac2 and Rac3 regulate numerous cellular processes. Since the cellular functions of Rac3 have not been defined, constitutively active V12Rac3 was used to identify targets that transduce its signals. We here identify human NRBP as a Rac family-interacting protein. NRBP formed a complex with activated Rac3. NRBP contains a kinase-homology domain and exhibits an associated kinase activity. NRBP represents a novel family of evolutionarily conserved proteins with homologs in C. elegans, D. melanogaster, mouse and human. Overexpression of NRBP in COS-1 cells failed to activate possible downstream targets of Rac3 including the JNK pathway, the p38 pathway or actin cytoskeletal rearrangements. Also, NRBP failed to co-localize with actin-based stress fibers or microspikes, or with the subcortical actin. However, overexpression of NRBP caused a dramatic redistribution of the Golgi-associated marker p58 to more peripheral locations within the cell, consistent with an impairment of the ER to Golgi transport. Immunocytochemistry showed that NRBP and activated Rac3 co-localized to endomembranes and at the cell periphery in lamellipodia. These results suggest that NRBP functions in subcellular trafficking and may be directed to specific subcellular locations through interaction with small GTPases of the Rho family.

Retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury.

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/beta(2) microglobulin (beta(2)M)(null) mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA(+) cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, <0.005% of enzymatically dispersed lung cells from NOD/SCID mice were CMFDA(+), which increased 10- to 100-fold in NOD/SCID/beta(2)M(null) mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA(+) cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.

Development of lentiviral vectors with regulated respiratory epithelial expression in vivo.

Development of gene transfer vectors with regulated, lung-specific expression will be a useful tool for studying lung biology and developing gene therapies. In this study we constructed a series of lentiviral vectors with regulatory elements predicted to produce lung-specific transgene expression: the surfactant protein C promoter (SPC) for alveolar epithelial type II cell (AECII) expression, the Clara cell 10-kD protein (CC10) for Clara cell expression in the airway, and the Jaagskiete sheep retrovirus (JSRV) promoter for expression in both cell types. Transgene expression from the SPC and CC10 vectors was restricted to AECII and Clara cell lines, respectively, while expression from the JSRV vector was observed in multiple respiratory and nonrespiratory cell types. After intratracheal delivery of lentivector supernatant to mice, transgene expression was observed in AECII from the SPC lentivector, and in Clara cells from the CC10-promoted lentivector. Transgene expression was not detected in nonrespiratory tissues after intravenous delivery of CC10 and SPC lentiviral vectors to murine recipients. In summary, incorporation of genomic regulatory elements from the SPC and CC10 genes resulted in respiratory specific transgene expression in vitro and in vivo. These vectors will provide a useful tool for the study of lung biology and the development of gene therapies for lung disorders.

Lentivirus vectors incorporating the immunoglobulin heavy chain enhancer and matrix attachment regions provide position-independent expression in B lymphocytes.

In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (E micro ) with and without associated matrix attachment regions (E micro MAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34(+) progenitors in vitro transduced with E micro - and E micro MAR-containing lentivectors. Lastly, we evaluated the expression from the E micro MAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the E micro and E micro MAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the E micro MAR-containing vector and not other cells types or vectors. Proviral genomes with the E micro MAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the E micro MAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.