Chimeric Cultures Research Articles

Assessing Human Airway Epithelial Progenitor Cells for Cystic Fibrosis Cell Therapy.

Cystic fibrosis (CF) is caused by loss-of-function mutations in the CFTR (CF transmembrane regulator) gene. Pharmacologic therapies directed at CFTR have been developed but are not effective for mutations that result in little or no mRNA or protein expression. Cell therapy is a potential mutation-agnostic approach to treatment. One strategy is to harvest human bronchial epithelial cells (HBECs) for gene addition or genetic correction, followed by expansion and engraftment. This approach will require cells to grow extensively while retaining their ability to reconstitute CFTR activity. We hypothesized that conditionally reprogrammed cell (CRC) technology, namely growth in the presence of irradiated feeder cells and a Rho kinase inhibitor, would enable expansion while maintaining cell capacity to express functional CFTR. Our goal was to compare expression of the basal cell marker NGFR (nerve growth factor receptor) and three-dimensional bronchosphere colony-forming efficiency (CFE) in early- and later-passage HBECs grown using nonproprietary bronchial epithelial growth medium or the CRC method. Cell number and CFTR activity were determined in a competitive repopulation assay employing chimeric air-liquid interface cultures. HBECs expanded using the CRC method expressed the highest NGFR levels, had the greatest 3D colony-forming efficiency at later passage, generated greater cell numbers in chimeric cultures, and most effectively reconstituted CFTR activity. In our study, the HBEC air-liquid interface model, an informative testing platform proven vital for the development of other CF therapies, illustrated that cells grown by CRC technology or equivalent methods may be useful for cell therapy of CF.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

Mosaic analysis of cell rearrangements during ureteric bud branching in dissociated/reaggregated kidney cultures and in vivo.

Cell rearrangements mediated by GDNF/Ret signaling underlie the formation of the ureteric bud (UB) tip domain during kidney development. Whether FGF signaling also influences these rearrangements is unknown. Chimeric embryos are a powerful tool for examining the genetic controls of cellular behaviors, but generating chimeras by traditional methods is expensive and laborious. Dissociated fetal kidney cells can reorganize to form complex structures including branching UB tubules, providing an easier method to generate renal chimeras. Cell behaviors in normal or chimeric kidney cultures were investigated using time-lapse imaging. In Spry1(-/-) ↔ wild-type chimeras, cells lacking Spry1 (a negative regulator of Ret and FGF receptor signaling) preferentially occupied the UB tips, as previously observed in traditional chimeras, thus validating this experimental system. In Fgfr2(UB-/-) ↔ wild-type chimeras, the wild-type cells preferentially occupied the tips. Independent evidence for a role of Fgfr2 in UB tip formation was obtained using Mosaic mutant Analysis with Spatial and Temporal control of Recombination (MASTR). Dissociation and reaggregation of fetal kidney cells of different genotypes, with suitable fluorescent markers, provides an efficient way to analyze cell behaviors in chimeric cultures. FGF/Fgfr2 signaling promotes UB cell rearrangements that form the tip domain, similarly to GDNF/Ret signaling.

Impaired development of cortico-striatal synaptic connectivity in a cell culture model of Huntington's disease

Huntington's disease (HD) is a genetically inherited neurodegenerative disease caused by a mutation in the gene encoding the huntingtin protein. This mutation results in progressive cell death that is particularly striking in the striatum. Recent evidence indicates that early HD is initially a disease of the synapse, in which subtle alterations in synaptic neurotransmission, particularly at the cortico-striatal (C-S) synapse, can be detected well in advance of cell death. Here, we used a cell culture model in which striatal neurons are co-cultured with cortical neurons, and monitored the development of C-S connectivity up to 21days in vitro (DIV) in cells cultured from either the YAC128 mouse model of HD or the background strain, FVB/N (wild-type; WT) mice. Our data demonstrate that while C-S connectivity in WT co-cultures develops rapidly and continuously from DIV 7 to 21, YAC128 C-S connectivity shows no significant growth from DIV 14 onward. Morphological and electrophysiological data suggest that a combination of pre- and postsynaptic mechanisms contribute to this effect, including a reduction in both the postsynaptic dendritic arborization and the size and replenishment rate of the presynaptic readily releasable pool of excitatory vesicles. Moreover, a chimeric culture strategy confirmed that the most robust impairment in C-S connectivity was only observed when mutant huntingtin was expressed both pre- and postsynaptically. In all, our data demonstrate a progressive HD synaptic phenotype in this co-culture system that may be exploited as a platform for identifying promising therapeutic strategies to prevent early HD-associated synaptopathy.

Regulation of System xc−Activity and Expression in Astrocytes by Interleukin‐1β

We recently demonstrated that interleukin-1β (IL-1β) increases system x(c)(-) (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system x(c)(-) subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT--the light chain that confers substrate specificity--in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system x(c)(-) inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system x(c)(-) activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.

Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR

Human amniotic fluid stem cells (hAFSCs) can be grown in large quantities, have a low risk for tumour development and harbour a high differentiation potential. They are a very promising new fetal stem cell type for cell-based therapy approaches and for studying differentiation processes without raising the ethical concerns associated with embryonic stem cells. Recently, a protocol for studies on renal development has been established in which murine embryonic kidneys are dissociated into single-cell suspension and then reaggregated to form organotypic renal structures. Using this approach, we formed chimeric renal structures via mixing murine embryonic kidney cells with monoclonal hAFSCs. We demonstrate here that hAFSCs harbour the potential to contribute to renal tissue formation accompanied by induction of specific renal marker expression. As part of the two kinase complexes mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signalling pathway, which is involved in the regulation of differentiation and in the development of a wide variety of human genetic diseases many with characteristic kidney symptoms. Modulating endogenous mTOR activity via specific siRNA approaches revealed that contribution of hAFSCs to renal tissue formation is regulated by mTORC1 and mTORC2. These findings (i) demonstrate renal differentiation potential of hAFSCs, (ii) prove chimeric cultures of mixtures of murine embryonic kidney cells and hAFSCs to be a powerful tool to study the effects of gene knockdowns for renal structure formation and (iii) provide new insights into the role of the mTOR pathway for renal development.

System xc−Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of hypoxic-ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1beta mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1beta-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of L-cystine, suggesting a role for the cystine/glutamate antiporter (System x(c)-). Indeed, dual System x(c)-/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1beta as wild-type cultures, an effect prevented by System x(c)-/mGluR1 antagonism. Finally, assessment of System x(c)- function and kinetics in IL-1beta-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI(+/+) neurons plated on top of IL-1RI(-/-) astrocytes, highlighting the importance of astrocyte-mediated alterations in System x(c)- as a novel contributor to the development and progression of hypoxic neuronal injury.

Lentiviral Rescue of Vascular Endothelial Growth Factor Receptor-2 Expression in Flk1−/− Embryonic Stem Cells Shows Early Priming of Endothelial Precursors

The vascular endothelial growth factor (VEGF) family and its receptors are important for vascular development and maintenance of blood vessels, as well as for angiogenesis, the formation of new vessels. Loss of VEGF receptor-2 (VEGFR-2; designated Flk-1 in mouse) results in arrest of vascular and hematopoietic development in vivo. We used lentiviral transduction to reconstitute VEGFR-2 expression in flk1-/- embryonic stem (ES) cells. VEGF-induced vasculogenesis and sprouting angiogenesis were rescued in transduced ES cultures differentiating in vitro as EBs. Although the transgene was expressed in the pluripotent stem cells and lacked linage restriction during differentiation, the extent of endothelial recruitment was similar to that in wild-type EBs. Reconstitution of VEGFR-2 in flk1-/- ES cells allowed only precommitted precursors to differentiate into functional endothelial cells able to organize into vascular structures. Chimeric EB cultures composed of wild-type ES cells mixed with flk1-/- ES cells or reconstituted VEGFR-2-expressing ES cells were created. In the chimeric cultures, flk1-/- endothelial precursors were excluded from wild-type vessel structures, whereas reconstituted VEGFR-2-expressing precursors became integrated together with wild-type endothelial cells to form chimeric vessels. We conclude that maturation of endothelial precursors, as well as organization into vascular structures, requires expression of VEGFR-2. Disclosure of potential conflicts of interest is found at the end of this article.

Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion

Malformations in secondary palate fusion will lead to cleft palate, a common human birth defect. Palate fusion involves the formation and subsequent degeneration of the medial edge epithelial seam. The cellular mechanisms underlying seam degeneration have been a major focus in the study of palatogenesis. Three mechanisms have been proposed for seam degeneration: lateral migration of medial edge epithelial cells; epithelial-mesenchymal trans-differentiation; and apoptosis of medial edge epithelial cells. However, there is still a great deal of controversy over these proposed mechanisms. In this study, we established a [Rosa26<-->C57BL/6] chimeric culture system, in which a Rosa26-originated ;blue' palatal shelf was paired with a C57BL/6-derived ;white' palatal shelf. Using this organ culture system, we observed the migration of medial edge epithelial cells to the nasal side, but not to the oral side. We also observed an anteroposterior migration of medial edge epithelial cells, which may play an important role in posterior palate fusion. To examine epithelial-mesenchymal transdifferentiation during palate fusion, we bred a cytokeratin 14-Cre transgenic line into the R26R background. In situ hybridization showed that the Cre transgene is expressed exclusively in the epithelium. However, beta-galactosidase staining gave extensive signals in the palatal mesenchymal region during and after palate fusion, demonstrating the occurrence of an epithelial-mesenchymal transdifferentiation mechanism during palate fusion. Finally, we showed that Apaf1 mutant mouse embryos are able to complete palate fusion without DNA fragmentation-mediated programmed cell death, indicating that this is not essential for palate fusion in vivo.

N-Cadherin Transsynaptically Regulates Short-Term Plasticity at Glutamatergic Synapses in Embryonic Stem Cell-Derived Neurons

The cell adhesion molecule N-cadherin has been proposed to regulate synapse formation in mammalian central neurons. This is based on its synaptic localization enabling alignment of presynaptic and postsynaptic specializations by an adhesion mechanism. However, a potential role of N-cadherin in regulating synaptic transmission has remained elusive. In this paper, a functional analysis of N-cadherin knock-out synapses was enabled by in vitro neuronal differentiation of mouse embryonic stem cells circumventing the early embryonic lethality of mice genetically null for N-cadherin. In our in vitro system, initial synapse formation was not altered in the absence of N-cadherin, which might be attributable to compensatory mechanisms. Here, we demonstrate that N-cadherin is required for regulating presynaptic function at glutamatergic synapses. An impairment in the availability of vesicles for exocytosis became apparent selectively during high activity. Short-term plasticity was strongly altered with synaptic depression enhanced in the absence of N-cadherin. Most intriguingly, facilitation was converted to depression under specific stimulation conditions. This indicates an important role of N-cadherin in the control of short-term plasticity. To analyze, whether N-cadherin regulates presynaptic function by a transsynaptic mechanism, we studied chimeric cultures consisting of wild-type neocortical neurons and ES cell-derived neurons. With N-cadherin absent only postsynaptically, we observed a similar increase in short-term synaptic depression as found in its complete absence. This indicates a retrograde control of short-term plasticity by N-cadherin. In summary, our results revealed an unexpected involvement of a synaptic adhesion molecule in the regulation of short-term plasticity at glutamatergic synapses.

Heparan Sulfate in trans Potentiates VEGFR-Mediated Angiogenesis

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

Taurolidine: preclinical evaluation of a novel, highly selective, agent for bone marrow purging.

Taurolidine has been shown to have remarkable cytotoxic activity against selected human tumor cells at concentrations that spare normal cells. In this study we have extended this observation and assessed the ability of Taurolidine to purge tumor cells from chimeric mixtures of bone marrow (BM) and neoplastic cells. Normal murine BM and human leukemic (HL-60) or ovarian (PA-1) tumor cell lines were used as models. Exposure of tumor cells to 2.5 mM Taurolidine for 1 h resulted in the complete elimination of viable cells. In contrast, exposure of BM to 5 mMTaurolidine for 1 h reduced CFU-GM, BFU-E and CFU-GEEM colony formation by only 23.0%, 19.6% and 25.2%, respectively. Inhibition of long-term BM culture (LTBMC) growth following a 1 h exposure to 5 mM Taurolidine also was approximately 20% compared to untreated LTBMC. Finally, chimeric cultures were generated from BM and HL-60GR or PA-1GR cells (tumor cells transfected with the geneticin resistance gene). Exposure of these chimeric cultures to 5 mM Taurolidine for 1 h totally eliminated viable cancer cells while minimally reducing viable BM cells. This finding was confirmed by subsequent positive selection for surviving tumor cells with geneticin. These findings reveal that Taurolidine holds promise for use in BM purging.

Permanent skin replacement using chimeric epithelial cultured sheets comprising xenogeneic and syngeneic keratinocytes.

The present study was undertaken to evaluate the possibility of permanent skin replacement using chimeric xenogeneic-syngeneic graftable sheets previously obtained in vitro. Newborn (<3 days old) BALB/c and human keratinocytes were isolated and cocultured in different ratios as follows: 50% BALB/c to 50% human and 25% BALB/c to 75% human keratinocytes. Four to 5 days after culture and prior to their grafting, all chimeric sheets contained both cell types in ratios similar to those used to seed the initial chimeric cultures. Fourteen and 30 days after chimeric sheet grafting onto BALB/c mice dorsum, the newly generated cutaneous tissue showed a histologically well-organized epidermis presenting basal and suprabasal cell layers. Cutaneous cells in these structures secreted laminin and type IV collagen in blood vessels, and at ground level of the dermoepidermal junction there were signs of physiologically active skin. Cell phenotyping revealed the presence of only syngeneic keratinocytes, whereas xenogeneic cells were passively eliminated without a total rejection of the chimeric implant. This selective and passive elimination of xenogeneic keratinocytes went through cellular and humoral immunity activation. Data suggest that this chimeric culture method can be used for cutaneous therapies such as large congenital nevi, skin ulcers, and extensively burned skin. Indeed, for large third-degree wounded skin treatment, this culture method may shorten the time (4-5 weeks) needed for cell growth and graftable sheet production. Moreover, since the ultimate aim in allogeneic and xenogeneic transplantation is to achieve an immunological acceptance and tolerance to these foreign tissues, the chimeric culture approach may provide ways to lighten tolerance phenomena on cutaneous tissue.

Transdifferentiation of putative neuronal cells of neural retina into lens: A demonstration by chick-quail chimeric cultures.

To elucidate the cell-type origin of lens cells, which differentiate in stationary cultures of neural retina, chimeric cultures between chick and quail cells were made to recombine xenoplastically the different cell fractions separated from 8- to 9-day cultures of 3.5-day-old embryonic neural retinal cells by means of centrifugation in Percoll. Extensive lentoidogenesis occurred in the recombination of the N2-fraction (consisting mostly of small round cells) with the E-fraction (containing a number of flattened epithelial cells). Taking advantage of the difference in electrophoretic mobility of chick and quail δ-crystallin, it was shown that this lens-specific protein, synthesized in the chimeric cultures, was mostly of the species-specificity of N2. Microscopic observations of histological sections of cell sheets of quail N2- and chick E-fraction chimeric cultures revealed that most cells with δ-crystallin, as identified by means of immunohistological detection, are provided with a nuclear marker characteristic of quail. By determining the level of activity of choline acetyltransferase and by examining the stainability with a fluorescent dye (Merocyanine-540), it was suggested that cells in the N2-fraction are primitive neuroblast-like cells. Thus, we can conclude that putative neuronal cells in early cultures of avian embryonic neural retina can transdifferentiate into lens cells.