A Comparison between Real-Time Quantitative PCR and DNA Hybridization for Quantitation of Male DNA following Myoblast Transplantation

Bosio, Erika; Lee-Pullen, Tracey F.; Fragall, Clayton; Beilharz, Manfred W.; Bennett, Alayne L.; Grounds, Miranda D.; Hodgetts, Stuart I.; Sammels, Leanne M.

doi:10.3727/000000004783983369

Cited by 9 publications

(5 citation statements)

References 13 publications

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“…loss of long-term expression of the marker in surviving cells (146,203,256), leakage of marker and subsequent uptake by host cells, toxicity from chronic exposure of markers, change in cellular distribution over time, and uptake/redistribution by scavenging host microglia/macrophages. Other markers may circumvent these factors by the use of species-specific markers, markers associated with a specific gene promoter or adenoviral (AdV), adeno-associated viral (AAV), retroviral (RV), and lentiviral (LV) vectors for the introduction of foreign genes encoding either the bacterial marker enzyme β-galactosidase/LacZ or green fluorescent protein (GFP), fluorescent (usually rhodamine or fluorescein) dye-coated latex microspheres/beads/nanospheres, and combined analysis with other assays such as IHC, retrograde-labeling, or EM, FISH, or real-time quantitative PCR using donor-specific sequences [e.g., Y1 DNA of male cells transplanted into female hosts (34,134), as well as the use of thymidine, bromodeoxyuridine (BrdU), and ethynyl deoxyuridine (EDU) analogues (133)].…”

Section: Ipsc-based Therapies As a Candidate For Sci Repairmentioning

confidence: 99%

Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury

et al. 2013

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Transplantation therapies aimed at repairing neurodegenerative and neuropathological conditions of the central nervous system (CNS) have utilized and tested a variety of cell candidates, each with its own unique set of advantages and disadvantages. The use and popularity of each cell type is guided by a number of factors including the nature of the experimental model, neuroprotection capacity, the ability to promote plasticity and guided axonal growth, and the cells' myelination capability. The promise of stem cells, with their reported ability to give rise to neuronal lineages to replace lost endogenous cells and myelin, integrate into host tissue, restore functional connectivity, and provide trophic support to enhance and direct intrinsic regenerative ability, has been seen as a most encouraging step forward. The advent of the induced pluripotent stem cell (iPSC), which represents the ability to "reprogram" somatic cells into a pluripotent state, hails the arrival of a new cell transplantation candidate for potential clinical application in therapies designed to promote repair and/or regeneration of the CNS. Since the initial development of iPSC technology, these cells have been extensively characterized in vitro and in a number of pathological conditions and were originally reported to be equivalent to embryonic stem cells (ESCs). This review highlights emerging evidence that suggests iPSCs are not necessarily indistinguishable from ESCs and may occupy a different "state" of pluripotency with differences in gene expression, methylation patterns, and genomic aberrations, which may reflect incomplete reprogramming and may therefore impact on the regenerative potential of these donor cells in therapies. It also highlights the limitations of current technologies used to generate these cells. Moreover, we provide a systematic review of the state of play with regard to the use of iPSCs in the treatment of neurodegenerative and neuropathological conditions. The importance of balancing the promise of this transplantation candidate in the light of these emerging properties is crucial as the potential application in the clinical setting approaches. The first of three sections in this review discusses (A) the pathophysiology of spinal cord injury (SCI) and how stem cell therapies can positively alter the pathology in experimental SCI. Part B summarizes (i) the available technologies to deliver transgenes to generate iPSCs and (ii) recent data comparing iPSCs to ESCs in terms of characteristics and molecular composition. Lastly, in (C) we evaluate iPSC-based therapies as a candidate to treat SCI on the basis of their neurite induction capability compared to embryonic stem cells and provide a summary of available in vivo data of iPSCs used in SCI and other disease models.

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Section: Ipsc-based Therapies As a Candidate For Sci Repairmentioning

confidence: 99%

Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury

et al. 2013

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“…As human TTN gene (367 exons, localized on 2q31) is present as only one single copy in the cell, it has been chosen to permit a direct correlation with the number of cells. Selecting a gene located on an autosomal chromosome, instead of using a probe recognising Y chromosome [15], extends our approach to human cells, isolated from both male and female subject. To ensure that our primers recognize exclusively genomic DNA sequences, we designed the couple of primers in the intron 1 of the human gene.…”

Section: Resultsmentioning

confidence: 99%

Combined methods to evaluate human cells in muscle xenografts

et al. 2019

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Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell- and gene- based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space. These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

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“…Several studies have been reported using RT-PCR to detect donor cells in transplantation models 9–10 . Dr. Schwarzenberger’s group first developed a murine bone marrow transplantation model using real-time PCR to amplify y-chromosome-specific 8 .…”

Section: Discussionmentioning

confidence: 99%

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Kang

2013

JoVE

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SHORT ABSTRACT Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions. LONG ABSTRACT Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

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A Comparison between Real-Time Quantitative PCR and DNA Hybridization for Quantitation of Male DNA following Myoblast Transplantation

Cited by 9 publications

References 13 publications

Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury

Systematic Review of Induced Pluripotent Stem Cell Technology as a Potential Clinical Therapy for Spinal Cord Injury

Combined methods to evaluate human cells in muscle xenografts

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

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