Survival and function of intrastriatally grafted primary fibroblasts genetically modified to produce L-dopa.

A combination of gene transfer and intracerebral grafting may provide a powerful technique for examining the role of discrete substances in the development or functioning of the brain. In the present study, primary fibroblasts obtained from a skin biopsy from inbred Fischer rats were used as donor cells for genetic modification and grafting. When grafted to the striatum of Fischer rats with a prior 6-hydroxydopamine lesion, primary fibroblasts containing a transgene for either tyrosine hydroxylase (TH) or beta-galactosidase survived for 10 weeks and continued to express the transgene. TH synthesized by the implanted fibroblasts appeared to convert tyrosine to L-dopa actively, as observed in vitro, and to affect the host brain, as assessed through a behavioral measurement. These results suggest that primary fibroblasts genetically altered to express TH have the capacity to deliver L-dopa locally to the striatum in quantities sufficient to compensate partially for the loss of intrinsic striatal dopaminergic input.     

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.     

Primary Skin Fibroblasts as a Model of Parkinson's Disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues.     

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease¹. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms² and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson''s disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.     

Donor age reflects the replicative lifespan of human fibroblasts in culture

Human fibroblasts, which have a finite lifespan in cultures, have been widely used as a model system for cellular aging, and frequently used as one model of human aging. But whether cellular aging contributes to organismal aging has been controversial. To reinvestigate this question, we cultured human fibroblasts from the skin of one individual volunteer collected at different ages. Over a period of 27 years (donor age 36 years to 62 years), we obtained skin cells four times at appropriate intervals, and established eight fibroblast lines. These human fibroblasts have presented evidence for a correlation between donor age and proliferative lifespan in vitro . This result parallels the fact that telomeric DNA size cultured fibroblasts decrease with the increase in donor age. These cell lines had a normal diploid human chromosome constitution and will be useful in studies of human biology including aging.     

A phosphatidylinositol transfer protein alpha-dependent survival factor protects cultured primary neurons against serum deprivation-induced cell death

Selective neuronal loss is a prominent feature in both acute and chronic neurological disorders. Recently, a link between neurodegeneration and a deficiency in the lipid transport protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it may be of importance that fibroblasts overexpressing PI-TPalpha are known to produce and secrete bioactive survival factors that protect fibroblasts against UV-induced apoptosis. In the present study it was investigated whether the conditioned medium of cells overexpressing PI-TPalpha (CMalpha) has neuroprotective effects on primary neurons in culture. We show that CMalpha is capable of protecting primary, spinal cord-derived motor neurons from serum deprivation-induced cell death. Since the conditioned medium of wild-type cells was much less effective, we infer that the neuroprotective effect of CMalpha is linked (in part) to the PI-TPalpha-dependent production of arachidonic acid metabolites. The neuroprotective activity of CMalpha is partly inhibited by suramin, a broad-spectrum antagonist of G-protein coupled receptors. Western blot analysis shows that brain cortex and spinal cord express relatively high levels of PI-TPalpha, suggesting that the survival factor may be produced in neuronal tissue. We propose that the bioactive survival factor is implicated in neuronal survival. If so, PI-TPalpha could be a promising target to be evaluated in studies on the prevention and treatment of neurological disorders.     

Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Skin Allograft Survival

Pretransplant donor lymphocyte infusion (DLI) has been shown to enhance donor-specific allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored donor cell subsets after DLI in a murine MHC class I L^d-mismatched skin transplantation model. We found that donor B cells, but not DCs, are the major surviving donor APCs in recipients following DLI. Infusing donor B, but not non-B, cells resulted in significantly enhanced donor-specific skin allograft survival. Furthermore, mice that had received donor B cells showed higher expression of Ly6A and CD62L on antigen-specific TCRαβ⁺CD3⁺CD4⁻CD8⁻NK1.1⁻ double negative (DN) regulatory T cells (Tregs). B cells presented alloantigen to DN Tregs and primed their proliferation in an antigen-specific fashion. Importantly, DN Tregs, activated by donor B cells, showed increased cytotoxicity toward anti-donor CD8⁺ T cells. These data demonstrate that donor B cells can enhance skin allograft survival, at least partially, by increasing recipient DN Treg-mediated killing of anti-donor CD8⁺ T cells. These findings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specific immune therapy to enhance allograft survival.     

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.     

Donor B cells in transplants augment clonal expansion and survival of pathogenic CD4+ T cells that mediate autoimmune-like chronic graft-versus-host disease

We reported that both donor CD4(+) T and B cells in transplants were required for induction of an autoimmune-like chronic graft-versus-host disease (cGVHD) in a murine model of DBA/2 donor to BALB/c recipient, but mechanisms whereby donor B cells augment cGVHD pathogenesis remain unknown. In this study, we report that, although donor B cells have little impact on acute GVHD severity, they play an important role in augmenting the persistence of tissue damage in the acute and chronic GVHD overlapping target organs (i.e., skin and lung); they also markedly augment damage in a prototypical cGVHD target organ, the salivary gland. During cGVHD pathogenesis, donor B cells are activated by donor CD4(+) T cells to upregulate MHC II and costimulatory molecules. Acting as efficient APCs, donor B cells augment donor CD4(+) T clonal expansion, autoreactivity, IL-7Rα expression, and survival. These qualitative changes markedly augment donor CD4(+) T cells' capacity in mediating autoimmune-like cGVHD, so that they mediate disease in the absence of donor B cells in secondary recipients. Therefore, a major mechanism whereby donor B cells augment cGVHD is through augmenting the clonal expansion, differentiation, and survival of pathogenic CD4(+) T cells.     

Effects of ambient oxygen concentration on the growth and antioxidant defenses of of human cell cultures established from fetal and postnatal skin

Oxygen toxicity is believed to arise from changes in the rates at which cells generate reactive oxygen species (ROS). Sensitivity to hyperoxia has been postulated to depend on levels of antioxidant defense. Human cells obtained from fetal tissues have lower antioxidant defenses than those obtained from adult tissue. The present study was performed to determine whether the differences in fetal and adult antioxidant defense levels modulated their responses to changes in the ambient oxygen concentration. Our results demonstrate that oxygen modulates the proliferation of human fetal and adult skin fibroblasts in a similar fashion. In general, skin fibroblasts grew better at approximately 31 mm Hg, regardless of donor age. Manganese superoxide dismutase, catalase, and glutathione peroxidase activities were lower in fetal cells than in adult fibroblasts. Copper/zinc superoxide dismutase and glucose-6-phosphate dehydrogenase were similar in fetal and postnatal tissues and were unaltered appreciably by hyperoxic exposure. Glutathione concentration increased at higher oxygen tensions; however, the increase was much greater in fetal cells than in cultures derived from adult skin. These observations demonstrate that the capacity of fetal and adult cells to cope with oxidative stress, while similar, result from distinct mechanisms.     

Inhibition of protein geranylgeranylation induces apoptosis in synovial fibroblasts

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-α-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA.     

Periostin localizes to cells in normal skin, but is associated with the extracellular matrix during wound repair

Epidermal tissue repair represents a complex series of temporal and dynamic events resulting in wound closure. Matricellular proteins, not normally expressed in quiescent adult tissues, play a pivotal role in wound repair and associated extracellular matrix remodeling by modulating the adhesion, migration, intracellular signaling, and gene expression of inflammatory cells, pericytes, fibroblasts and keratinocytes. Several matricellular proteins show temporal expression during dermal wound repair, but the expression pattern of the recently identified matricellular protein, periostin, has not yet been characterized. The primary aim of this study was to assess whether periostin protein is present in healthy human skin or in pathological remodeling (Nevus). The second aim was to determine if periostin is expressed during dermal wound repair. Using immunohistochemistry, periostin reactivity was detected in the keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin. In pathological nevus samples, periostin was present in the extracellular matrix. In excisional wounds in mice, periostin protein was first detected in the granulation tissue at day 3, with levels peaking at day 7. Periostin protein co-localized with α-smooth muscle actin-positive cells and keratinocytes, but not CD68 positive inflammatory cells. We conclude that periostin is normally expressed at the cellular level in human and murine skin, but additionally becomes extracellular during tissue remodeling. Periostin may represent a new therapeutic target for modulating the wound repair process.     

Translational aspects of cardiac cell therapy

Cell therapy has been intensely studied for over a decade as a potential treatment for ischaemic heart disease. While initial trials using skeletal myoblasts, bone marrow cells and peripheral blood stem cells showed promise in improving cardiac function, benefits were found to be short‐lived likely related to limited survival and engraftment of the delivered cells. The discovery of putative cardiac ‘progenitor’ cells as well as the creation of induced pluripotent stem cells has led to the delivery of cells potentially capable of electromechanical integration into existing tissue. An alternative strategy involving either direct reprogramming of endogenous cardiac fibroblasts or stimulation of resident cardiomyocytes to regenerate new myocytes can potentially overcome the limitations of exogenous cell delivery. Complimentary approaches utilizing combination cell therapy and bioengineering techniques may be necessary to provide the proper milieu for clinically significant regeneration. Clinical trials employing bone marrow cells, mesenchymal stem cells and cardiac progenitor cells have demonstrated safety of catheter based cell delivery, with suggestion of limited improvement in ventricular function and reduction in infarct size. Ongoing trials are investigating potential benefits to outcome such as morbidity and mortality. These and future trials will clarify the optimal cell types and delivery conditions for therapeutic effect.     

Therapeutic use of limbal stem cells

Stem cells are defined as relatively undifferentiated cells that have the capacity to generate more differentiated daughter cells. Limbal stem cells are responsible for epithelial tissue repair and regeneration throughout the life. Limbal stem cells have been localized to the Palisades of Vogt in the limbal region. Limbal stem cells have a higher proliferative potential compared to the cells of peripheral and central cornea. Limbal stem cells have the capacity to maintain normal corneal homeostasis. However, in some pathological states, such as chemical and thermal burns, Stevens-Johnson syndrome, and ocular pemphigoid limbal stem cells fail to maintain the corneal epithelial integrity. In such situations, limbal stem cell transplantation has been required as a therapeutic option. In unilateral disorders, the usual source of stem cells is the contralateral eyes, but if the disease is bilateral stem cell allografts have to be dissected from family members or cadaver eyes. The advent of ex vivo expansion of limbal stem cells from a small biopsy specimen has reduced the risk of limbal deficiency in the donor eye. Concomitant immunosuppressive therapy promotes donor-derived epithelial cell viability, but some evidences suggest that donor-derived epithelial stem cell viability is not sustained indefinitely. Thus, long-term follow-up studies are required to ascertain whether donor limbal stem cell survival or promotion of recolonization by resident recipient stem cells occurs in restored recipient epithelium. However, this is not an easy task since a definitive limbal stem cell marker has not been identified yet. This review will discuss the therapeutic usage of limbal stem cells in the corneal epithelial disorders.     

Survival and DNA Repair of Somatic Cell Hybrids after Ultraviolet Irradiation

THE technique of somatic cell hybridization has opened up studies on genetic regulation¹ and human genetic analysis^2–5. Hybrid cells are isolated in conditions that select against parental cells while allowing hybrids to survive by genomic complementation. In xeroderma pigmentosum (XP), a human disease with an autosomal recessive defect in an early stage of DNA repair⁶, the skin is extremely sensitive to sunlight in vivo⁷ and skin fibroblasts show sharply reduced survival following ultraviolet irradiation in vitro^8,9. This communication concerns the use of ultraviolet irradiation in combination with a chemical method to produce hybrids between fibroblasts from XP and a hamster line, followed by analysis of these cells for their capacity to survive and repair DNA after exposure to ultraviolet. Methods for initiation and propagation of skin fibroblasts from two subjects, male and female siblings with XP, have been described⁸. Details on the origin of the TG2 line of golden hamster fibroblasts, which has a non-reverting mutation in the gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the general hybridization procedure¹⁰ and methods for cell survival and DNA repair by unscheduled synthesis⁸ were also described previously. Hybrids were produced by fusion with Sendai virus and selected by ultraviolet irradiation followed by culture on HAT medium (Fig. 1).     

Pigeon liver phosphoprotein phosphatase: an effective activator of pyruvate dehydrogenase in tissue homogenates

A fluoride-insensitive, non-metal-requiring pyruvate dehydrogenase phosphatase has been purified 730-fold from pigeon liver acetone powder and proven to be a convenient reagent for studies of pyruvate dehydrogenase complex and its activation (phosphorylation) state in brain and other tissues. This phosphatase is a cytoplasmic enzyme (Mr = 80,000), and fits the functional definition of a type 1 phosphoprotein phosphatase. The pigeon liver phosphatase can be used to activate pyruvate dehydrogenase complex in vitro in brain and other crude tissue homogenates. Addition of the cytoplasmic pigeon liver phosphatase to a homogenate from rat or mouse brain frozen in situ activated pyruvate dehydrogenase to levels comparable to that found in ischemic brain. The fluoride insensitivity of this phosphatase was used to develop a convenient technique for stopping the pyruvate dehydrogenase activation state in situ in cultured skin fibroblasts and then fully activating the complex in vitro in 5 min. The use of this phosphatase as a reagent can facilitate the study of pyruvate dehydrogenase activation defects in mammalian tissues including cultured cells in normal and disease states.     

Immunological considerations for embryonic and induced pluripotent stem cell banking

Recent advances in stem cell technology have generated enthusiasm for their potential to study and treat a diverse range of human disease. Pluripotent human stem cells for therapeutic use may, in principle, be obtained from two sources: embryonic stem cells (hESCs), which are capable of extensive self-renewal and expansion and have the potential to differentiate into any somatic tissue, and induced pluripotent stem cells (iPSCs), which are derived from differentiated tissue such as adult skin fibroblasts and appear to have the same properties and potential, but their generation is not dependent upon a source of embryos. The likelihood that clinical transplantation of hESC- or iPSC-derived tissues from an unrelated (allogeneic) donor that express foreign human leucocyte antigens (HLA) may undergo immunological rejection requires the formulation of strategies to attenuate the host immune response to transplanted tissue. In clinical practice, individualized iPSC tissue derived from the intended recipient offers the possibility of personalized stem cell therapy in which graft rejection would not occur, but the logistics of achieving this on a large scale are problematic owing to relatively inefficient reprogramming techniques and high costs. The creation of stem cell banks comprising HLA-typed hESCs and iPSCs is a strategy that is proposed to overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. Estimates have shown that a stem cell bank containing around 10 highly selected cell lines with conserved homozygous HLA haplotypes would provide matched tissue for the majority of the UK population. These simulations have practical, financial, political and ethical implications for the establishment and design of stem cell banks incorporating cell lines with HLA types that are compatible with different ethnic populations throughout the world.     

Successful Survival of Grafted Transgenic Neural Plate Cells in Adult Central Nervous System Environment

1. Accumulating evidence indicates that damaged brain functions can be ameliorated in a variety of animal models by the grafting of fetal neuronal cell or tissue into damaged brain. Clinical trials are under way to determine whether human fetal mesencephalic tissue can ameliorate motor functions in patients with Parkinson's disease.2. Autopsy findings of parkinsonian patient implanted with human fetal mesencephalic tissue clearly revealed that the fetal neuronal graft can survive for an extended period of time in the human brain and densely reinnervate the surrounding host striatal tissue.3. It is, however, still important to obtain more practical, effective, and ethically justifiable donor material for the future clinical application of the procedures. Desirable properties for the donor cells include long-term survival in the brain, neuronal cell type for the reconstruction of damaged neural circuits, and susceptibility to genetic manipulation for the practical use.4. With the development of molecular biology techniques, genetic modification and transplantation of the donor neuronal cells might be a feasible way to cure many kinds of central nervous system diseases toward a graft-gene therapy.