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Protocol

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Medicine

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

Published: February 17th, 2019

DOI:

10.3791/58115

1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiology, Stanford University School of Medicine, 3Medical Service, Cardiology Section, Veteran Affairs Palo Alto Health Care System, 4Department of Pathology, Stanford University School of Medicine, 5Department of Radiology, Molecular Imaging Program, Stanford University School of Medicine, 6Peking University Shenzhen Health Science Institute, 7Department of Radiation Oncology, Stanford University School of Medicine

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of "stem cell tourism," the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.

External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

The development of stem cell therapies for tissue regeneration has encountered a number of barriers in the past several decades, hampering efforts for efficient clinical deployment. These hurdles include poor cell retention at sites of delivery, stem cell immunogenicity, and the neoplastic potential to form teratomas1. Tumorigenicity is of particular clinical concern as it can potentially harm stem cell transplant recipients2. Accounts of tumor formation due to unregulated stem cell injections have already been reported in multiple clinical settings3,4,

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This animal experiment was approved and performed under the Institutional Review Board and the Administrative Panel on Laboratory Animal Care at Stanford University.

1. Cell Culture of iPSCs

  1. Grow human iPSCs derived by lentiviral reprogramming on 6-well plates coated with basement membrane matrix (e.g., matrigel, referred to as matrix hereon).
  2. Daily change the media of the iPSCs with enriched culture medium (see Table of Materials) incubating at 37 &.......

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Injected mice typically will demonstrate teratoma growth formation after 4–8 weeks as confirmed by BLI imaging (Figure 2). Tumors will shrink dramatically when irradiated with a cumulative dose of 18 Gy given one month after cell delivery, resulting in a significant decrease in luciferase signal (Figure 2). Importantly, normal tissues taken 5 mm from the irradiated site do not appear to have any significant damage (

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Preclinical data and anecdotal cases from victims of "stem cell tourism" confirm that the risk of developing teratomas is a serious drawback associated with PSC treatments23. Development of careful approaches to prevent and treat the neoplastic risk associated with stem cell therapies is, therefore, an important step in facilitating the clinical translation of regenerative stem cell therapies. In this article, we described a method of therapeutic targeting of PSC-associated teratomas using.......

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The authors would like to thank the National Institutes of Health R01 HL134830 (PKN), K08 HL135343 (KS), and 5F32HL134221 (JWR); the Howard Hughes Medical Institute (ASL); and the Stanford Cardiovascular Institute (ASL) for their support.

....

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Name Company Catalog Number Comments
Induced Pluripotent Stem Cell Control Line Stanford University Nguyen Lab Cell culture of iPSC
Corning matrigel basement membrane matrix 354234 Fisher Scientific CB-40234 Cell culture of iPSC
Essential 8 culture medium ATCC-The global bioresource center 30-2203 Cell culture of iPSC
Tryple E Gibco 12605-036 Cell culture of iPSC
Y27632 inhibitor 2 HCL (ROCK Inhibitor) Fisher Scientific S104950MG Cell culture of iPSC
Lentivirus Cyagen P170721-1001cjn Transduction of iPSC with double fusion reporter gene
Polyrbrene Infection/Transfection Reagent Millipore Sigma TR-1003-G Transduction of iPSC with double fusion reporter gene
Fluc-eGFP reporter gene driven by ubiquitin promoter Stanford University Sam Gambhir lab Transduction of iPSC with double fusion reporter gene
D-luciferin Perkin Elmer 122799 Transduction of iPSC with double fusion reporter gene and BLI
Flow cytometer (BD FACSARIA III) BD Biosciences  FACSAria Transduction of iPSC with double fusion reporter gene
microplate spectrofluorometer (Glomax Navigator System) Promega Bio Systems, Sunnyvale, CA GM2000 Transduction of iPSC with double fusion reporter gene
Xenogen IVIS 200  Perkin Elmer 124262 BLI
Isoflurane Sigma-Aldrich CDS019936 irradiation
X-Rad SmART image-guided irradiator  Precision X-ray Inc., North Branford, CT X-Rad SmART irradiation
RT_Image software package Stanford University (http://rtimage.sourceforge.net/) RT_Image v0.2β Irradiation

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