In vivo telomere dynamics of human hematopoietic stem cells

Aging in vivo and cell division in vitro are associated with telomere shortening. Several lines of evidence suggest that telomere length may be a good predictor of the long term replicative capacity of cells. To investigate the natural fate of chromosome telomeres of hematopoietic stem cells in vivo, we measured the telomere length of peripheral blood granulocytes from 11 fully engrafted bone marrow transplant recipients and from their respective donors. In 10 of 11 donor–recipient pairs, the telomere length was significantly reduced in the recipient and the extent of reduction correlated inversely with the number of nucleated cells infused. These data provide internally controlled in vivo evidence that, concomitantly with their proliferation, hematopoietic stem cells lose telomere length; it is possible that, as a result, their proliferative potential is reduced. These findings must be taken into account when developing new protocols in which few stem cells are used for bone marrow transplantation or for gene therapy.     

X-ray-induced mutations in mouse embryonic stem cells

Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here. First, a highly polymorphic, germ-line competent F_1(129/Sv-+^Tyr₊^p _{× CAST/Ei)} mouse embryonic stem cell line was established. Then, x-ray and UV-induced mutagenesis was performed to determine the feasibility of generating deletion complexes throughout the mouse genome. Reported here are the selection protocols, induced mutation frequencies, cytogenetic and extensive molecular analysis of mutations at the X-chromosome-linked hypoxanthine phosphoribosyltransferase (Hprt) locus and at the neural cell adhesion molecule (Ncam) locus located on chromosome 9. Mutation analysis with PCR-based polymorphic microsatellite markers revealed deletions of <3 cM at the Hprt locus, whereas results consistent with deletions covering >28 cM were observed at the Ncam locus. Fluorescence in situ hybridization with a chromosome 9 paint revealed that some of the Ncam deletions were accompanied by complex chromosome rearrangements. In addition, deletion mapping in combination with loss of heterozygosity of microsatellite markers revealed a putative haploinsufficient region distal to Ncam. These data indicate that it is feasible to generate x-ray-induced deletion complexes in mouse embryonic stem cells.     

Myofibrillogenesis visualized in living embryonic cardiomyocytes

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, α-actinin. The expression of this fluorescent protein provided an in vivo label for structures containing α-actinin. The GFP–α-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed de novo at the spreading edges of cardiomyocytes, contained punctate concentrations of α-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form de novo near the cell membrane are premyofibrils—i.e., the precursors of mature myofibrils.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cell transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho^+/++ (dull/bright) and Rho⁻ (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho^+/++ cell population contained >99% of committed progenitor cells with in vitro colony-forming ability. The Rho⁻ cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho⁻ stem cells or purified Rho⁻ stem cells in combination with large numbers of Rho^+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho⁻ stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract

Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF⁶⁵ in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.     

Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo

Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis. Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo. The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture. Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo. Transfected cells were detected both in the surface epithelium and in submucosal glands. In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.     

Antigen presentation in retroviral vector-mediated gene transfer in vivo

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte/macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vector-immunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.     

Ethanolamine modulates the rate of rat hepatocyte proliferation in vitro and in vivo

A low molecular weight, heat-resistant hepatotrophic factor in an extract from the bovine intestinal mucosa was purified and identified as ethanolamine by structural analyses. The mode of action of ethanolamine in vitro and in vivo coincided with that of the crude extract of the tissue, indicating that ethanolamine is the active component. Ethanolamine synergistically elevated the stimulation of DNA synthesis in hepatocytes in primary culture when added together with a growth factor, such as epidermal growth factor, with the ED₅₀ being 20 μM, although it showed little stimulatory effect by itself. Contrary to these in vitro results, the intraperitoneal administration of ethanolamine hydrochloride (24 mg of ethanolamine per kg of body weight) enhanced hepatocyte proliferation in regenerating rat livers after two-thirds hepatectomy without the administration of any growth factors. In the regenerating liver, hepatocyte proliferation may be initiated by an endogenous growth factor, but the supply of ethanolamine in circulation may not be sufficient for optimal hepatocyte proliferation; thus, the exogenous administration of ethanolamine may further enhance hepatocyte proliferation. Ethanolamine in circulation may be a humoral hepatotrophic factor.     

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas⁺ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas⁺ Renca tumor by inducing cell death, whereas the elimination of Fas⁻ CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas⁺ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.     

Protamine-Cre recombinase transgenes efficiently recombine target sequences in the male germ line of mice, but not in embryonic stem cells

The production of subtle or conditional mutations in mice through the combined use of site-specific and homologous recombination has become an increasingly widespread experimental paradigm in mammalian genetics. Embryonic stem cells containing recombinase transgenes that were expressed in the male germ line, but not in other tissues or in the embryonic stem cells themselves, would substantially simplify the production of such alleles. Here we show that transgenes comprised of the mouse protamine 1 promoter and the Cre recombinase coding sequence mediate the efficient recombination of a Cre target transgene in the male germ line, but not in other tissues. Embryonic stem cell lines generated from one of these transgenic strains were transfected with targeting vectors that included loxP-flanked selectable markers, and homologously recombined alleles containing the marker and functional loxP sites were isolated. These results establish the potential of the system for substantially reducing the time, effort, and resources required to produce homologously recombined alleles in mice that have been secondarily rearranged by a site-specific recombinase.     

Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF⁶⁵ to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF⁶⁵ to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF⁶⁵ can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.     

High affinity ligands from in vitro selection: Complex targets

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems.     

Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34⁺ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34⁺ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34⁺ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34⁺ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

A major objective of recent HIV research is to develop a “cure” for this virus infection that avoids lifelong adherence to antiretroviral therapy (ART). One of the approaches toward reaching this objective has been to genetically delete or mutate genes encoding for proteins that promote HIV infection and spread. An attractive candidate for this strategy is the Ccr5 gene, for which a genetic mutation causing a 32-bp deletion has been shown to be associated with natural protection from HIV infection and disease (1, 2). The Ccr5 gene encodes CCR5, a human cell-surface chemokine receptor that is a coreceptor for HIV attachment and infection of cells (3, 4). The Ccr5 allele with its 32-bp deletion results in a truncated isoform of the CCR5 receptor, CCR5Δ32, which is not expressed at the cell surface. Thus, entry of the virus into the cell is blocked (5).Induced pluripotent stem (iPS) cells (6), because of their capacity to differentiate into CD34⁺ hematopoietic stem cells (HSCs) (7), can reconstitute a full immune system (8, 9). These iPS cells are therefore a target of choice for genetic engineering. Our group and others have demonstrated that iPS cells generated from the peripheral blood mononuclear cells (PBMC) of both healthy individuals (10) and HIV-infected patients under ART (11) can have their wild-type allele of the Ccr5 gene genetically edited to carry the Ccr5 Δ32 mutation (12, 13). Notably, using CRISPR/Cas9 technology, the Ccr5 gene can be modified to have the naturally occurring Δ32 variant allele that has been associated with resistance to R5-tropic viruses. Moreover, while it is not present at the cell surface, the truncated CCR5Δ32 protein is still expressed and, as such, could have other important physiological roles (14–17).We have confirmed that the genetically modified Ccr5 Δ32 iPS cells can be differentiated into CD34⁺ HSCs in vitro (10, 18). Under appropriate cell culture conditions, they can give rise to various myeloid and lymphoid cell lineages (10, 11, 18). This result can also be observed with the formation of teratomas following the injection of large quantities of iPS cells into mice. Teratomas are multicellular tumors composed of many different cell types including HSCs. Notably, immune cells with the CCR5Δ32 mutation differentiated in vitro from the genetically modified iPS cell-derived HSCs and inoculated with HIV are resistant to R5-tropic virus infection (10, 18).These results have suggested that editing Ccr5 in iPS cells from HIV-infected subjects can be a promising strategy toward an HIV cure. The pluripotent stem cells can be induced from a small number of PBMC from the patients and genetically modified to become resistant to HIV infection (10, 11, 18). In this case, leukapheresis to obtain large amounts of these cells (19) is not required. The edited HSCs could then be transplanted back to the original patient without concern for immune cell rejection. Therefore, because these experiments were performed in cell culture, an important remaining question is whether in vitro-edited iPS cells can differentiate into HSCs that can be transplanted back into a recipient in vivo (20).To address this question, transplantation of the in vitro-derived CD34⁺ cells was attempted under various conditions in animal models of humanized or immunodeficient mice (21). In approaches to obtain sufficient numbers of CD34⁺ cells for transplantation, our ability to grow them in vitro offered an opportunity. However, although we could expand CD34⁺ cells substantially in culture (18), we observed that engraftment of these cell culture-derived CD34⁺ cells in humanized NSG-BLT mice did not occur. Thus, alternatively, to study the genetically edited cells in vivo, we explored the use of differentiated CD34⁺ cells in vivo via the generation of teratomas from iPS cells. We found that not only did these teratomas successfully yield human CD34⁺ cells, but importantly, these CD34⁺ cells could engraft in recipient immunodeficient NSG mice. This observation has been made by Nakauchi and colleagues (22) with different mouse strains. Finally, we confirmed that the PBMC formed in mice from these teratoma-derived genetically edited CD34⁺ cells are resistant to ex vivo R5-tropic HIV infection when they carry the mutant Δ32 Ccr5 allele.     

An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherin-positive, and ZO-1-positive by immunostaining) and BSN cells, which are largely mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an in vitro tubulogenesis system. BSN cells expressed hepatocyte growth factor (HGF) and transforming growth factor-β1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early in vitro branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor α, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens in vitro.