Joyce Axelman

Effect of ageing on reactivation of the human X-linked HPRT locus.

In mammals, X-chromosome dosage compensation is achieved by inactivating one X chromosome in female cells1. To test the hypothesis2 that genes on the silent X chromosome reactivate as a consequence of ageing, we examined the X-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 41 women who are heterozygous for mutations at this locus, leading to severe deficiency of the enzyme (Lesch-Nyhan syndrome3). We find that heterozygotes who are more than 10 yr old have an excess of HPRT+ skin fibroblast clones (59% rather than the 50% expected as a consequence of random X inactivation) but this excess does not increase with age. Further studies of eight of these heterozygotes show that the silent locus does not detectably reactivate spontaneously in culture, but only in response to treatment with 5-aza-2-deoxycytidine, a potent inhibitor of methylation. There is no age difference in the frequency of this reactivation as assayed by HATr clones, and a more sensitive autoradiographic assay shows only a twofold difference between young and old heterozy-gotes. Thus, age-related reactivation is not a feature of all X-linked loci, and may have species, tissue and locus-specific determinants.

Photoactivated Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans

Photoactivated composite poly(ethylene glycol)–hyaluronic acid biomaterials demonstrate enhanced physicochemical properties for facial soft tissue reconstruction. Photogenic Polymers Can Fix the Flaws Some people just love the spotlight; apparently, some polymers do too. Here, Hillel et al. introduce a class of composite polymers that react favorably to light by crosslinking within minutes. These polymers, composed of synthetic poly(ethylene glycol) (PEG) and natural hyaluronic acid (HA), have been developed for reconstructing facial soft tissue. Deformities in craniofacial soft tissue are a clinical challenge because even small defects can have a major impact on a person’s social behavior and psychological well-being. Hillel and colleagues created a polymeric composite that can be injected into the damaged site, massaged into shape, and then crosslinked in situ with light. A transdermal light exposure method would allow clinicians to inject a liquid polymer, rather than surgically inserting already-polymerized material. First, the authors designed an array of light-emitting diodes to penetrate up to 4 mm of human skin (both light and dark) without any painful side effects. A 2-min exposure to light was enough to crosslink the PEG-HA material under the skin. Next, the polymer was tailored to closely match the elastic properties of native soft tissues, such as human fat. Various amounts of PEG and concentrations of HA were tested, with the authors arriving at an optimal combination of 100 mg PEG and 24 mg/ml HA. When polymerized subcutaneously in rats, the PEG-HA implants were able to maintain near their original volume for up to 491 days, whereas control HA injections were completely resorbed. Notably, these HA-based materials were partially reversible with the addition of the enzyme hyaluronidase. To translate this material to the clinic, Hillel et al. then tested the PEG-HA composites in humans. The polymer was injected into the intradermal space in the abdomen of three patients scheduled to undergo abdominoplasty surgery. Similar to the rodent studies, the PEG-HA material persisted for 12 weeks, whereas the control HA injections lost their shape. An inflammatory response was observed surrounding the injections. It is clear that this new photo-friendly polymer and transdermal crosslinking method will be clinically useful for soft tissue reconstruction—perhaps even encouraging more people to put their best faces forward in the spotlight. Soft tissue reconstruction often requires multiple surgical procedures that can result in scars and disfiguration. Facial soft tissue reconstruction represents a clinical challenge because even subtle deformities can severely affect an individual’s social and psychological function. We therefore developed a biosynthetic soft tissue replacement composed of poly(ethylene glycol) (PEG) and hyaluronic acid (HA) that can be injected and photocrosslinked in situ with transdermal light exposure. Modulating the ratio of synthetic to biological polymer allowed us to tune implant elasticity and volume persistence. In a small-animal model, implanted photocrosslinked PEG-HA showed a dose-dependent relationship between increasing PEG concentration and enhanced implant volume persistence. In direct comparison with commercial HA injections, the PEG-HA implants maintained significantly greater average volumes and heights. Reversibility of the implant volume was achieved with hyaluronidase injection. Pilot clinical testing in human patients confirmed the feasibility of the transdermal photocrosslinking approach for implantation in abdomen soft tissue, although an inflammatory response was observed surrounding some of the materials.

Genetic complementation after fusion of Tay-Sachs and Sandhoff cells

THE N-acetyl-β-D-glucosaminidase (hexosaminidase) activity in cultured human fibroblasts consists of at least three components (A, B and C)1–3. At least two of these (designated Hex A and Hex B) seem to be closely related, for (1) antiserum against Hex A reacts against Hex B, and vice versa4,5 (2) in Tay-Sachs disease only Hex A activity is deficient, accompanied by an increase in Hex B activity in certain tissues1,6, and (3) both Hex A and Hex B activities are missing in Sandhoff disease, another autosomal recessive disorder7. But in spite of several theories8–11, the precise relationship between these components remains unknown, as does the nature of the genetic and biochemical relationships between the two diseases. To investigate these questions we have fused Tay-Sachs with Sandhoff fibroblasts and obtained cultures containing heterokaryons which produce a hexosaminidase which is absent from the parent lines. It has the electrophoretic and heat lability characteristics of the Hex A found in normal fibroblasts.

Reactivation of X-linked genes in human fibroblasts transformed by origin-defective SV40

To determine if expression of genes on the inactive X is inducible in human cells, we looked for reactivation events in a clone of fibroblasts transformed with origin-defective SV40. The karyotype of these cells was grossly heteroploid so that the aneuploidy associated with SV40 transformation occurs even in the absence of viral replication. This transformed clone, heterozygous for hypoxanthine phosphoribosyltransferase (HPRT), lacks HPRT activity, as the mutant allele is on the active X and the normal allele on the inactive X. Reactivation of the HPRT+allele on the inactive X was observed at a frequency of 6 x 10−5per cell and increased approximately eightfold following treatment with the cytidine analogs 5-azacytidine (5azaC) and 5-azadeoxycytidine. The fact that spontaneous reactivation is detectable in some clones, but not all, suggests that the environment of the SV40-transformed cell, although not sufficient to induce generalized derepression, increases the frequency of rare reactivation events. The methylation pattern at the HPRTlocus revealed transformation-associated alterations that may have predisposed these cells to reactivation events, spontaneous as well as 5azaC-induced.

Imprinting Status of GαS, NESP55, and XLαs in Cell Cultures Derived from Human Embryonic Germ Cells: GNAS Imprinting in Human Embryonic Germ Cells

GNAS is a complex gene that through use of alternative first exons encodes signaling proteins Gαs and XLαs plus neurosecretory protein NESP55. Tissue‐specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune‐Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of Gαs, NESP55, and XLαs prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of Gαs was biallelic (maternal allele: 52.6%± 2.5%; paternal allele: 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%± 10%; paternal allele: 18.1%± 10%; p= 0.002) and XLαs was preferentially expressed from the paternal allele (maternal allele: 2.7%± 0.3%; paternal allele: 97.3%± 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5–11 weeks postfertilization, and that imprinting of XLαs occurs very early postfertilization. By contrast, mprinting of Gαs most likely occurs after 11 weeks postfertilization and after tissue differentiation.

Validation of a small animal model for soft tissue filler characterization.

Background Rigorous preclinical testing of soft tissue fillers has been lacking. No animal model has emerged as an accepted standard to evaluate tissue filler longevity. Objective To validate a small animal model to compare soft tissue filler degradation and tissue reaction. Methods Preliminary experiments compared caliper with magnetic resonance imaging volumetric analysis. Next, four hyaluronic acid (HA) fillers were injected into the dermis of Sprague–Dawley rats. The three dimensions of the implants were measured at day 0, day 1, and monthly for 1 year or complete resorption of the filler. Volumetric, histologic, and statistical analyses were performed. Results Magnetic resonance imaging results validated caliper‐based volumetric measurements. Histology demonstrated injections in the subcutaneous space just deep to the dermis and panniculus carnosus. High‐ and very high‐concentration HA fillers maintained significantly greater volumes and volume ratios than low‐concentration HA fillers throughout the duration of the study. Conclusions The rat subcutis model demonstrated the ability to differentiate between HA fillers with different residence times. The caliper‐based rat‐subcutis method demonstrated consistent volumetric analysis and correlated with human residence times of HA fillers. These quantitative results validate the rat subcutis model as an expedited preclinical model for HA fillers.

Derivation and Differentiation of Human Embryonic Germ Cells

Publisher Summary Embryonic germ (EG) cells are pluripotent stem cells derived from primordial germ cells (PGCs) that arise in the late embryonic and early fetal period of development. Embryonic stem (ES) cells were first derived from the inner cell mass of mouse pre-implantation embryos, and EG cells were initially derived from mouse PGCs. Subsequently, EG cells have been derived from chicken, pig, and human PGCs. Pig, chicken, and mouse EG cells have been demonstrated to contribute to experimentally produced chimeric animals, including germline transmission in the latter two species. Human EG cells can be derived from PGCs by using methods similar to those used to derive mouse EG cultures. Like mouse embryonic stem and EG cells, human EG cells require leukemia inhibitory factor (LIF) for proliferation as undifferentiated stem cells. Unlike mouse EG cells, however, human EG cells do not readily lose their dependence on exogenous cytokines and factors supplied by the feeder layer, and they have a higher frequency of spontaneous differentiation into embryoid bodies (EBs). Although EBs are a loss to the pluripotent stem cell population, they are a source of cells expressing markers of mature cellular phenotypes, as well as their presumed progenitors and precursors. Cells that retain a high capacity for cell proliferation and express makers of multiple lineages can be isolated from EBs, and can be used in a variety of in vitro and in vivo differentiation paradigms. The current challenges are to match individual EB-derived (EBD) cultures to desired endpoints, and to enrich or purify populations of cells within EBD cultures to more specifically address biological requirements.

MOLECULAR CHARACTERIZATION OF A DELETED X CHROMOSOME (XQ13.3-XQ21.31) EXHIBITING RANDOM X INACTIVATION

As a result of selection following random X chromosome inactivation in human females, X chromosomes with visible deletions are usually inactive in every somatic cell. We have studied a female with mental retardation and dysmorphic features whose karyotype includes an X chromosome with a visible interstitial deletion in the proximal long arm. Based on cytogenetic analysis, the proximal breakpoint appeared to be in band Xq13.1, and the distal one in band q21.3. However, molecular analyses show that less of the q13 band is missing than cytogenetic studies indicated, as the deletion includes only loci from the region Xq13.3 to Xq21.31. Unexpectedly, studies of chromosome replication show that the pattern of X inactivation is random. Whereas the deleted X chromosome is late replicating in some cells from all tissues studied, it is early replicating in the majority of blood lymphocytes and skin fibroblasts, and is the active X chromosome in many of the hybrids derived from skin fibroblasts. As this chromosome is able to inactivate, it must include those DNA sequences from the X-inactivation center (XIC) that are essential forcis X inactivation. Molecular studies show that the XIC region, at Xq13.2, is present, so it is unlikely that the lack of consistent inactivation of this chromosome is attributable to close proximity of the breakpoint to the XIC. Supporting this conclusion is the similarity of the breakpoints to those of the other chromosomes we studied, whose deletions clearly do not interfere with the ability to inactivate. Our results show that deletions distal to DXS441 in Xq13.2 do not interfere withcis X inactivation. We attribute the random pattern of X inactivation reported here to the fact that in the tissues studied, cells with this interstitial deletion are not at a selective disadvantage.

DERIVATION OF PLURIPOTENT STEM CELLS FROM CULTURED HUMAN PRIMORDIAL GERM CELLS

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.