The Science of Human Fetal Tissue Research

 

A robust ethical dialogue about human fetal tissue (HFT) and its use in biomedical research requires a strong grasp of the scientific and practical details of this issue. Here, we provide detailed descriptions of the types of tissues collected as well as how and why they are used in laboratory experimentation and sometimes clinical application, focusing on current and future research directions. We also identify various materials derived from HFT, particularly cells that are isolated and cultured to establish cell strains and lines. We distinguish procedures that rely on the ongoing destruction of human life compared to those that do not. We consider the conscience concerns of scientists, physicians, patients, and the public, especially in light of poor transparency and the interdependent nature of scientific work. All of these elements are important when discussing various aspects of ethics, legislation, regulation, and funding.

Human Development

In order to better understand human fetal tissue research, one must first have a basic understanding of human prenatal development and the motivations for some scientists to acquire the remains of the unborn who have died in utero. Conception, or fertilization, is the union of a human egg and sperm, resulting in a single-cell human embryo (or zygote). At fertilization, a new human life is created. This single-cell embryo contains all the unique genetic information necessary for this new human being, exhibits self-directed development, and meets the biological definition of an organism.[1] The developing human organism is called an embryo from the time of fertilization until the end of the eighth week of gestation. It is then called a fetus until birth, around 38 weeks post-fertilization (or 40 weeks gestation). During development, cells in the early embryo replicate and differentiate to make many types of human cells, which will eventually form all the tissues and organs of the human body.

The earliest epiblast cells, present within the inner cell mass of a five-day-old embryo blastocyst, are the progenitors for all the cell types (>200 types) present in the fully developed human body. Scientists can extract pluripotent epiblast cells and use them to derive human embryonic stem cells (hESCs), which can become almost any cell type through a process called differentiation. Human embryonic stem cells are distinct from fetal tissue and fetal cell lines and strains. Fetal tissues and fetal cells are further along in the cell development process than hESCs, with varying degrees of differentiation depending on the organ of origin and the gestational age of the fetus.

Human Embryonic and Fetal Tissues

In the context of the continuum of human development, prenatal human cells and tissues are used as research materials in three major ways: (1) intact embryos are used for reproductive research like genome editing, chimeras, cloning, and 3-parent embryos; (2) embryos in the blastocyst phase are disrupted to collect hESCs; and (3) deceased fetuses are dissected to collect tissue and cells of interest. In the case of (3), the gestational age is sometimes early enough that the human being is still in the embryonic stage (before nine weeks gestation). For simplicity, tissues from both embryos and fetuses are classified together as “human fetal tissue.”

In (1) and (2), the embryo in vitro is deliberately destroyed at some point after experimentation or during stem cell extraction. In (3), the embryo or fetus used for experimentation is already deceased. Death may occur by either natural or unnatural causes. In natural cases of prenatal demise, there is no deliberate harm done to the unborn child (i.e. spontaneous abortion, also known as miscarriage or stillbirth). In unnatural cases, the deliberate act of an induced abortion kills the unborn child. A recent review provides an in-depth treatment of the scientific, legal, and ethical issues surrounding the use of HFT from elective abortions in research.[2] Here the focus is specifically on how tissues from embryos and fetuses (classified together as “HFT”) are currently used in scientific research.

HFT consists of organs, tissues, or cells taken directly from an unborn child. While it is possible to collect such tissue from the unborn who have died of natural causes, this is not common practice; HFT research almost always involves using an embryo or fetus who died from an induced abortion. Although obtaining whole organs can be difficult with dilation and evacuation abortion procedures, it is possible to obtain at least parts of the requested organs, such as brain, liver, kidney, lung, skin, and thymus. The scientific literature includes multiple reports of methods to improve sample quality and mitigate problems with tissue fragmentation and timing of tissue collection.[3] In some cases, the abortion procedure is altered to maintain organ/tissue integrity, which poses ethical concerns regarding informed consent as well as legal implications.[4]

Justification for Using Fetal Tissue in Research

Experiments using HFT date back about 100 years. Some of these experiments yielded promising results, but others failed to produce positive outcomes or faced significant technical problems. Additionally, some resulted in serious complications once they reached clinical trials.[5]

Scientists have argued that, compared to tissues obtained from adult donors, HFT contains a greater number of immature cells that are highly proliferative, less immunogenic, and more flexible, meaning that these less specialized cells can be more readily grown and manipulated phenotypically in culture. With this rationale, HFT and cells derived from HFT (e.g., fetal stem cells, tissue-specific cells) are often described as unique and valuable materials that cannot be replaced by other cell types.[6] The failures of HFT transplantation, some of them catastrophic to patients, have moved researchers away from transplanting fetal tissue.[7] However, HFT and the cell lines and strains derived from it are still widely used as model systems for both physiological and pathological studies, as well as in the testing and production of some pharmaceutical products.[8]

The physiological and phenotypic properties of HFT and cells derived from it are not equivalent to those of mature adult human tissues. Yet, the majority of research projects using HFT and its derivatives are focused on modeling, studying, and/or treating conditions that occur in mature adult tissues. These biological differences, while known and well understood, are often not acknowledged, leading to inherent design flaws in many studies that can limit success and applicability. Indeed, such research would benefit greatly from more appropriate models available today that do not involve the termination of human fetuses.

Primary HFT Research Applications

Primary HFT consists of organs, tissues, or cells collected directly from the deceased unborn child. There are multiple research applications that make direct use of this tissue. The organs or tissues themselves may be used, or these may be separated into their component cells to be kept alive in culture for a limited amount of time without producing successive generations of cells; these are known as primary cells. In some cases, specific cell types are isolated and cultured, such as stem cells. All research using primary HFT requires ongoing tissue collection since each sample can only be used for a limited number of experiments. Currently, this primary HFT is almost always obtained from an abortion.

Alternatively, the cells present in some HFT samples can be cultured and multiplied in the laboratory, and successive generations of cells from a single abortion can be mass produced and used for decades for a variety of applications. These are known as fetal cell lines and cell strains and will be discussed further below. Both of these categories are distinct from primary tissue and primary cells.[9]

Primary HFT collected immediately from the body of an intentionally aborted fetus is transferred to research labs to conduct science experiments including (1) observational or analytical studies (e.g., histology, genomics, proteomics); (2) generation of tissue transplant mice with a human immune system (e.g. bone marrow, liver, thymus, or “BLT,” humanized mice); (3) clinical transplantation; and (4) isolation of non-immortal cells with a limited lifespan (i.e., primary cells, stem cells). Fetal tissue supply is limited, and the ongoing destruction of human life is necessary to meet continued experimental demands. Some examples are explained in more detail below.

Observational and Analytical Studies

One major research area that uses primary HFT is “fate mapping,” in which different types of fetal tissue (e.g., brain, heart, or eye tissue) may be analyzed, often at various developmental stages, for expression of specific molecules (DNA, RNA, protein, etc.). For example, Li et al. collected bulk tissue and cells from several aborted fetal brains starting at seven weeks gestational age for their genomic study; Kim et al. created a draft map of the human proteome using several human tissues, including fetal heart, liver, gut, ovaries, testis, and brain.[10] A human cell atlas of fetal gene expression was recently published using 15 different human fetal organs.[11]

In the area of infectious disease, researchers used brains obtained from aborted fetuses (15 to 24 weeks gestational age) in order to study the mechanism of Zika infection in the second-trimester human developing brain.[12] In the area of eye disease and disorders of vision, a grant currently funded by the National Institutes of Health (NIH) proposes to “label green and red cone cells in human . . . fetal eyes using an RNA in situ hybridization technique that successfully distinguishes green and red opsin expression.”[13]

Some researchers have used human fetal scalps for growing human skin and hair follicles.[14] Epidermal and dermal cells are isolated in order to examine their ability to graft into a mouse and regenerate into mature skin (one phenotype of mature skin being hair growth).

In a 2014 paper, scalps from eight different fetuses, 15 to18 weeks gestation, were obtained from Advanced Bioscience Resources (ABR) in Alameda, California.[15] A more recent study engrafted human fetal skin tissue into immunodeficient rodents to produce a model that could provide a means for studying skin infections.[16]

Human Immune System Mice

Human Immune System (HIS) mice (also known as humanized mice) are a common application for human fetal tissue research. HIS mice are an important laboratory model for many areas of study, including infectious disease, immunology, oncology, and pharmacology. Mice are bred and/or genetically engineered to be immunodeficient (i.e., to have little or no mouse-specific immune function)[17] to introduce a human-like immune system by engrafting human cells. HFT is one possible source of these human cells; other sources, meeting higher ethical standards, include cord blood, bone marrow, peripheral blood mononuclear cells, spleen cells, lymph node cells,[18] or neonatal thymus removed during heart surgeries.[19] This approach endeavors to overcome innate interspecies differences in infectivity, immune response, and/or disease progression in order to study human-specific pathogens and diseases in rodent models,[20] such as HIV/AIDS, tuberculosis, Dengue, Epstein-Barr virus, and cancer. HIS mice are also used to study potential treatments for infectious disease, such as Truvada for HIV.[21] The bone marrow/liver/thymus “BLT” mouse model is the major application for primary HFT in this field. Human fetal liver and thymus (16 to 22 weeks gestation) are dissected and implanted under the kidney capsule of immunodeficient mice, along with an injection of CD34+ blood stem cells from the remaining liver and/or bone marrow of the same fetus into the mouse tail vein.[22]

Two newer HIS models have also been described using fetal lung tissue to create the human lung-only mouse (LoM) and the bone marrow/liver/thymus/lung “BLT-L” mouse. The LoM and BLT-L HIS mouse models were developed for studying human pathogen infection, analyzing human immune response, and testing antiviral drug protection against viral infection.[23] A more recent 2021 study specifically examined SARS-CoV-2 infection, the virus that causes COVID-19, using the LoM model.[24] Lung tissue from at least 12 abortions (likely 16 to 22 weeks gestation) was used to create 78 lung-only HIS mice. Human lung tissue was purchased from ABR. The fetal lung tissue was dissected into sections, and two pieces were implanted onto the back of each mouse. Mice inherently cannot be infected with coronavirus; they have a different version of ACE2 (the viral receptor target) that is not compatible with SARS-CoV-2 infection. Human ACE2 from the fetal tissue provided the right receptor target for infection to occur.

However, ethical alternatives to such research are available as described in the report by Sander Lee and Prentice. In this particular study, one ethically acceptable option would be to use transgenic mice that express human ACE2 and thus can be infected with SARS-CoV-2 for COVID-19 studies without the use of fetal tissues.[25] Another alternative is to use hamsters because their ACE2 receptor is genetically more similar to human ACE2 and has been shown to be compatible for SARS-CoV-2 infection.[26]

The “NeoThy mouse” is another non-fetal model that recapitulates many desirable properties of the BLT mouse, with the advantages of higher efficiency, lower cost, less variability, and higher relevance to patients in the clinic. Thymus tissue is obtained from neonatal cardiac surgeries, with informed consent, and implanted into immunodeficient mice, with concurrent injection of hematopoietic stem cells from cord blood.[27]

Clinical Transplantation

In the case of transplantation, no clinical trials using HFT have been federally funded since 2005. Attempts to treat diabetes, Parkinson’s disease, Huntington’s disease, cancer, hematopoietic disorders, metabolic disorders, immunodeficiencies, and other disorders by HFT transplantation have had varied, lackluster, and sometimes dangerous results.[28] Notably, as many as 24 fetuses, of gestational age 16 to 20 weeks, have been used per diabetes patient, with less than 2% of patients responding to this treatment.[29] For Parkinson’s disease, up to six fetuses (6 to 12 weeks gestation) have been used per patient.[30] In 2001, the first full report of a clinical trial was published,[31] and the doctors described to the New York Times the “devastating” side effects some patients experienced—writhing, twisting, and jerking uncontrollably.[32] In 2003, a large, controlled study reported potentially disabling tremors in over half of the patients.[33] Neither study showed benefit to patients, and a moratorium on HFT transplants for Parkinson’s disease patients ensued.

Transplantation of neural HFT for treatment of Parkinson’s disease and other neurological disorders have sometimes resulted in various “graft overgrowths” and non-brain tissues growing in patient’s brains, such as skin-like tissue, hair, cartilage, and other tissue nodules.[34] Furthermore, long-term follow-up with some Parkinson’s disease patients who received HFT transplants showed that even if the fetal tissue grafted successfully, it took on signs of disease.[35] Purified fetal stem cells (derived from HFT) can also have this effect, as seen in the case of a boy who developed tumors in his brain and spinal cord following fetal stem cell injections to treat ataxia telangiectasia.[36]

HFT-Derived Cells and Research Applications

There are two major forms of cells derived from HFT: finite cell strains and continuous cell lines. In both cases, primary tissue is dissociated into its component cells, which are then cultured in laboratory dishes with optimal conditions for proliferation.[37] Portions of cells from subsequent generations can be flash-frozen and stored for future use, shipped to laboratories around the world, and/or held by a repository or biotechnology company to be maintained and sold for decades to come. When thawed, they will continue to produce new generations of functional cells.

Thus, HFT collected from a single abortion can give rise to large numbers of laboratory-cultured cells that can be used for decades in labs around the world for a variety of purposes. They are bought and sold regularly, as are products derived from or produced by them (proteins, DNA, viruses, vaccines, etc.). These cell lines and strains can be patented and licensed. This is the most pervasive use of abortion-derived HFT in biomedical research and pharmaceutical production and thus presents a significant challenge to researchers, physicians, and patients who wish to avoid any connection to induced abortion. It should be noted that while a particular fetal cell line or strain originates from a single fetus, the discovery process often utilizes many aborted fetuses before achieving this goal.[38]

Fetal origin is not a requirement for generating either continuous cell lines or finite cell strains. The creators of the first fetal finite cell strains noted that adult human diploid cell alternatives existed at the time.[39] Today, adult stem cells, umbilical cord blood, and induced pluripotent stem cells, for example, provide additional advantages and are ethical alternatives for various research applications.[40]

Finite Cell Strains

Finite cell strains are genetically normal, or diploid, and thus have a limited lifespan. However, with the right cell type and careful culturing technique, they can survive and replicate exponentially for many generations before losing their utility for experimental and production purposes, such that “one could have cells available at any given time and in almost limitless numbers.”[41] These are also known as diploid, impermanent, or non-immortal cell strains. Examples derived from HFT include WI-38,[42] MRC-5,[43] and Walvax-2,[44] all of which are used for production of certain vaccines. WI-38 and MRC-5 are also widely used for laboratory research, as are IMR-90[45] and IMR-91.[46]

While research with existing fetal finite cell strains does not require collection of primary tissue from newly aborted fetuses, it can nevertheless contribute to demand for such tissue. Finite cell strains have a limited lifespan and thus need to be replaced or replenished eventually. Practical considerations add to this need, as not every single vial of cells will be nurtured to its full theoretical proliferation potential. In addition, access may be limited by the entities in possession and control of the cells (e.g., in order to ensure sufficient supply remains available for vaccine production), so some companies and researchers seek to create their own cell strains to use without interference. The most prominent example is the success of WI-38, developed in 1961 and still widely used today, which has motivated researchers to obtain abortion-derived HFT to create at least four additional fetal finite cell strains, as recently as 2015.[47]

Continuous Cell Lines

Continuous cell lines have been genetically altered by viruses, oncogenes, mutagens, or other agents to proliferate indefinitely; this process is called transformation or immortalization. They are genetically abnormal but can provide robust and reproducible systems to study cellular functions, as well as to produce proteins, DNA, and other products of interest. They are also known as immortalized, established, or permanent cell lines. Examples with human fetal origin include HEK293,[48] perhaps the most widely used fetal cell line in the world, and PER.C6.[49]

The most recognized and deeply entrenched continuous fetal cell line is HEK293. “HEK” stands for “human embryonic kidney.” HEK293 cells are sometimes known simply as 293 cells. In 1973, Graham et al. used fragments of adenovirus DNA to immortalize cells isolated from the primary kidney tissue of a female fetus[50] who was most likely aborted in the Netherlands in 1972.[51] HEK293 is now ubiquitous in biomedical research and industry, holding second place only to HeLa cells in cell biology research and to Chinese hamster ovary cells in biopharmaceutical production.[52] For some applications, like adenovirus studies,[53] viral vector propagation, and small-scale protein production,[54] they are first choice. These cells grow rapidly and easily; transfect (i.e., accept DNA or RNA) readily and with high efficiency; and produce both proteins and viruses in high yields. They have been modified to grow in suspension and in the absence of animal serum. All of these properties make them desirable for both research and manufacturing applications. Therapies manufactured using HEK293 include, but are not limited to, the gene therapy LUXTURNA to treat retinal dystrophy, CAR-T cell immunotherapy to treat specific types of blood cancer, and therapeutic proteins for hemophilia.

The names of the many cell lines derived from HEK293 may or may not indicate their fetal origin. Cellosaurus, an online knowledge resource on cell lines, lists 403 “children” for HEK293.[55] Furthermore, either the parent HEK293 or one of these many derivative cell lines may be packaged in commercially available kits, which may have trademarked names and no clear indication of fetal origin. Even if a researcher chooses a cell line with no fetal origin, it may have been genetically modified using vectors produced in HEK293, which may or may not be reported in the paperwork. This lack of transparency can make it difficult to know a cell line’s origin unless a researcher specifically seeks out this information—by tracing through the literature trail, searching online resources like Cellosaurus or the American Type Culture Collection website, and/or calling suppliers directly to inquire for more information (which they may or may not be able to provide). The provenance of cellular materials passed informally among colleagues can be even more difficult to discern depending on the quality of records kept and shared. Indeed, many of the details surrounding the origins of the HEK293 line itself have been lost to time.[56]

HEK293 (and its derivative cell lines) also suffer from multiple important scientific shortcomings. The viral transformation process inserted a large segment of adenoviral DNA into the cellular genome and also imparted an abnormal number of chromosomes as well as significant genetic heterogeneity and instability.[57] Furthermore, like many continuous cell lines, cultivation in a variety of laboratories over many years has led to genetic drift, making some experimental results difficult to reproduce in different labs.

As with finite cell strains, the success and widespread use of fetal continuous cell lines motivate researchers to create new lines with more desirable properties or to fit a specific research niche (such as a specific cell type to study a particular disease). Shortcomings of old transformation technologies become apparent, and scientists seek to improve the methods in future attempts. PER.C6 is a clear example of a fetal continuous cell line engineered to avoid certain pitfalls of HEK293 and to be suitable for pharmaceutical manufacturing;[58] it is also a proprietary product. Recent examples of fetal cells immortalized to model specific cell types include liver,[59] pancreatic beta cells,[60] and brain pericytes,[61] just to name a few.

Thus, the demand for primary HFT from newly aborted fetuses continues to this day in the area of cell line/strain derivation, fueled by the successful use of cells taken from fetuses who were terminated many years ago. Moreover, researchers are the end users who provide a market for commercialized fetal cell strains and lines.[62] Without legal and/or regulatory restrictions or other incentives (from consumers, industry, or the research community themselves) to use alternative sources of cells, these efforts will undoubtedly continue into the future. Thus, using materials derived from abortions of the past does have an ongoing impact on fetuses who are terminated in the present.

Conscience in the Laboratory

For scientists who wish to avoid the use of all human tissues and cells that are ethically dubious due to the nature of their procurement or origin, the status quo presents significant challenges. A thorough discussion of these challenges has previously been published.[63] In brief, conscientious objections may extend to primary HFT as well as any biological material derived from it, such as primary cell cultures, finite cell strains, continuous cell lines, DNA plasmids, viral vectors, and recombinant proteins. Questions also arise about using data and analysis tools generated by others through the use of these materials, including proteomic and genomic databases, for instance. The widespread and entrenched use of fetal cell lines like HEK293 and fetal cell strains like WI-38 often pose the greatest challenge to a scientist in this regard.

Scientists may be confronted with the use of any of these materials in their own labs, in their collaborators’ labs, in their colleagues’ labs, and in the papers and grants they review. In these cases, the scientist faces choices about varying levels of participation and support for such research. For subordinate lab members, such as graduate students, postdoctoral associates, or laboratory technicians—and even for junior faculty—power imbalance further impacts freedom of conscience. Lack of transparency regarding HFT-derived materials also compounds these difficulties.

It is worth noting that physicians and patients (i.e., all of us) also face some of these same dilemmas as they relate to medical treatments—such as biologic medicines, vaccines, and cell-based therapies—which may be produced, developed, and/or tested using abortion-derived materials. The recent debates over the ethics of vaccines for COVID-19 highlight these difficulties because some manufacturers still choose to continue using old fetal cell lines or strains from abortion for production and/or testing (i.e., HEK293, PER.C6, MRC-5), regardless of ethical alternatives currently available.

The nature of an individual’s culpability and connection to abortion when partaking in various research and consumer activities has been analyzed by others[64] and is also addressed elsewhere in this Special Report. There are differing ethical positions held and distinctions made regarding these materials. There are also differences in ethical positions on using HFT obtained following natural fetal demise (i.e., miscarriage or stillbirth).[65]

Conclusion

Cells and tissues from the bodies of human embryos and fetuses, deceased either by induced abortion or natural causes (although less common), are used in various aspects of biomedical research that range from basic observational and cell studies to the most common application of generating HIS mice. Primary HFT must be repeatedly obtained from deceased unborn children in order to continue the research.

HFT is also used to create finite cell strains, which have a normal diploid genome and a limited (though long) lifespan, as well as continuous cell lines, which are immortalized by genetic alteration. Both types of HFT-derived cells are cultured in the laboratory and used for many decades in a wide variety of applications, and their supply does not require additional abortions to take place.

The use of HFT, particularly when obtained from the deliberate destruction of the unborn child through the act of abortion, is controversial and has been fraught with ethical concerns. Ethical positions are varied and sometimes quite nuanced. Hence, we endeavor here to provide accurate and up-to-date scientific information to support the important and precise ethical discussions that are so crucial to moving science forward in a way that respects and serves human dignity.

As science continues to advance, one thing is certain. Some researchers will continue to explore ways to use aborted fetal tissue and its derivatives while they are allowed to do so or until the act of abortion is prohibited or significantly curtailed. Until then, it is important to have accurate information and an understanding of the factors that define HFT and its derivatives. In addition, there needs to be complete transparency by those in academic and industrial settings who choose to use HFT so that fellow scientists and the public are able to make fully informed decisions whether to accept or reject its use, as well as federal funding support, in science and medicine.

 

References


[1] Maureen L. Condic, “When Does Human Life Begin? A Scientific Perspective,” Westchester Institute White Paper Series 1, no. 1 (2008): 1–18, https://bdfund.org/wp-content/uploads/2016/05/wi_whitepaper_life_print.pdf.

[2] Tara Sander Lee et al., “Human Fetal Tissue from Elective Abortions in Research and Medicine: Science, Ethics, and the Law,” Issues in Law and Medicine 35, no. 1 (2020): 3–61.

[3] Jie Lu et al., “Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue,” Journal of Visualized Experiments (JoVE) 51, no. e2681 (2011): http://doi.org/10.3791/2681. A video of the procedure using human fetal brains is available at http://www.jove.com/details.php?id=2681. Bruno Gridelli et al., “Efficient Human Fetal Liver Cell Isolation Protocol Based on Vascular Perfusion for Liver Cell–Based Therapy and Case Report on Cell Transplantation,” Liver Transplantation 18, no. 2 (2012): 226–37, https://doi.org/10.1002/lt.22322; Nahid Waleh et al., “Patterns of Gene Expression in the Ductus Arteriosus Are Related to Environmental and Genetic Risk Factors for Persistent Ductus Patency,” Pediatric Research 68, no. 4 (2010): 292–97, https://dx.doi.org/10.1203%2FPDR.0b013e3181ed8609; Christopher Pekor et al., “Induction of Hepatic and Endothelial Differentiation by Perfusion in a Three-Dimensional Cell Culture Model of Human Fetal Liver,” Tissue Engineering Part C: Methods 21, no. 7 (2015): 705–15, https://doi.org/10.1089/ten.tec.2014.0453; Giada Pietrosi and Cinzia Chinnaci, “Report on Liver Cell Transplantation Using Human Fetal Liver Cells,” in Hepatocyte Transplantation: Methods and Protocols, ed. Peggy Stock and Bruno Christ (New York: Humana Press, 2017), https://doi.org/10.1007/978-1-4939-6506-9_20.

[4] See Grant and Kirtley’s articles for more information on informed consent, and Prentice’s article for more on legal implications.

[5] Articles in this Report by Grant, Kirtley, and Prentice outline some of these early studies.

[6] Sally Temple and Lawrence S. B. Goldstein, “Why We Need Fetal Tissue Research,” Science 363, no. 6424 (2019): 207, https://doi.org/10.1126/science.aaw6299.

[7] Gina Kolata, “Parkinson’s Research Is Set Back by Failure of Fetal Cell Implants,” The New York Times, March 8, 2001, http://www.nytimes.com/2001/03/08/health/08PARK.html.

[8] See the article by Sander Lee and Prentice for detailed discussion of non-controversial alternatives currently available for the most common HFT research applications.

[9] See also articles in this report by Grant and Prentice.

[10] Mingfeng Li et al., “Integrative Functional Genomic Analysis of Human Brain Development and Neuropsychiatric Risks,” Science 362, no. 6420 (2018): https://doi.org/10.1126/science.aat7615; Min-Sik Kim et al., “A Draft Map of the Human Proteome,” Nature 509, no. 7502 (2014): 575–81, https://doi.org/10.1038/nature13302.

[11] Junyue Cao et al., “A Human Cell Atlas of Fetal Gene Expression,” Science 370, no. 6518 (2020): 1–17, https://doi.org/10.1126/scienceaba7721.

[12] Hanna Retallack et al., “Zika Virus Cell Tropism in the Developing Human Brain and Inhibition by Azithromycin,” Proceedings of the National Academy of Sciences of the United States of America 113, no. 50 (2016): 14,408–13, https://doi.org/10.1073/pnas.1618029113.

[13] Sarah E. Hadyniak, “Specification of Green and Red Cone Cells in the Human Eye: Project Details,” Project Number 1F31EY029157-01A1, NIH, accessed August 2, 2021, https://reporter.nih.gov/project-details/9835305#details.

[14] Xunwei Wu et al., “Full-Thickness Skin with Mature Hair Follicles Generated from Tissue Culture Expanded Human Cells,” Tissue Engineering: Part A 20, no. 23–24 (2014): 3314–21, https://doi.org10.1089/ten.TEA.2013.0759; Ying Zheng et al., “Mature Hair Follicles Generated from Dissociated Cells: A Universal Mechanism of Folliculoneogenesis,” Developmental Dynamics 239, no. 10 (2010): 2619–26, https://doi.org/10.1002/dvdy.22398.

[15] Wu et al., “Full-Thickness Skin with Mature Hair Follicles Generated from Tissue Culture Expanded Human Cells.”

[16] Yash Agarwal et al., “Development of Humanized Mouse and Rat Models with Full‑Thickness Human Skin and Autologous Immune Cells,” Scientific Reports 10, no. 14598 (2020): https://doi.org/10.1038/s41598-020-71548-z.

[17] “Why Humanized Mice?” The Jackson Laboratory, July 21, 2020, https://www.jax.org/news-and-insights/jax-blog/2020/july/why-humanized-mice#.

[18] Dörthe Masemann, Stephan Ludwig, and Yvonne Boergeling, “Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses,” International Journal of Molecular Science 21, no. 23 (2020): 9289, https://doi.org/10.3390/ijms21239289; Kylie Su Mei Yong, Zhisheng Her, and Qingfeng Chen, “Humanized Mice as Unique Tools for Human-Specific Studies,” Archivum Immunologiae Therapiae Experamentalis 66, no. 4 (2018): 245–66, https://doi.org/10.1007/s00005-018-0506-x.

[19] Matthew E. Brown et al., “A Humanized Mouse Model Generated Using Surplus Neonatal Tissue,” Stem Cell Reports 10, no. 4 (2018): 1175–83, https://doi.org/10.1016/j.stemcr.2018.02.011.

[20] Masemann, Ludwig, and Boergeling, “Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses.”

[21] Reviewed in Bradford K. Berges and Mark R. Rowan, “The Utility of the New Generation of Humanized Mice to Study HIV-1 Infection: Transmission, Prevention, Pathogenesis, and Treatment,” Retrovirology 8, no. 65 (2011): https://doi.org/10.1186/1742-4690-8-65.

[22] Y. Maurice Morillon et al., “The Development of Next-Generation PBMC Humanized Mice for Preclinical Investigation of Cancer Immunotherapeutic Agents,” Anticancer Research 40, no. 10 (2020): 5329–41, https://doi.org/10.21873/anticanres.14540; Yong, Her, and Chen, “Humanized Mice as Unique Tools for Human-Specific Studies.”

[23] Angela Wahl et al., “Precision Mouse Models with Expanded Tropism for Human Pathogens,” National Biotechnology 37, no. 10 (2019): 1163–73, https://doi.org/10.1038/s41587-019-0225-9.

[24] Angela Wahl et al., “SARS-CoV-2 Infection Is Effectively Treated and Prevented by EIDD-2801,” Nature 591, no. 7850 (2021): https://doi.org/10.1038/s41586-021-03312-w.

[25] Fatai S. Oladunni et al., “Lethality of SARS-CoV-2 Infection in K18 Human Angiotensin-Converting Enzyme 2 Transgenic Mice,” Nature Communications 11, no. 1 (2020): 6122, https://doi.org/10.1038/241467-020-19891-7.

[26] Jasper Fuk-Woo Chan et al., “Simulation of the Clinical and Pathological Manifestations of Coronavirus Disease 2019 (COVID-19) in a Golden Syrian Hamster Model: Implications for Disease Pathogenesis and Transmissibility,” Clinical Infectious Diseases 71, no. 9 (2020): 2428–46, https://doi.org/10.1093/cid/ciaa325.

[27] Brown et al., “A Humanized Mouse Model Generated Using Surplus Neonatal Tissue.”

[28] “History of Fetal Tissue Research and Transplants,” Charlotte Lozier Institute, July 27, 2015, https://lozierinstitute.org/history-of-fetal-tissue-research-and-transplants.

[29] K. Federlin, R. G. Bretzel, and B. J. Hering, “Recent Achievements in Experimental and Clinical Islet Transplantation,” Diabetic Medicine 8, no. 1 (1991): 5–12, https://doi.org/10.1111/j.1464-5491.1991.tb01508.x; reviewed in Alan Fine, “Transplantation of Fetal Cells and Tissue: An Overview,” Canadian Medical Association Journal 151, no. 9 (1994): 1261, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1337326/#.

[30] Fine, “Transplantation of Fetal Cells and Tissue,” 1261.

[31] C. R. Freed et al., “Transplantation of Embryonic Dopamine Neurons for Severe Parkinson’s Disease,” The New England Journal of Medicine 344, no. 10 (2001): 710, https://doi.org/10.1056/nejm200103083441002.

[32] Gina Kolata, “Parkinson’s Research Is Set Back by Failure of Fetal Cell Implants,” The New York Times, March 8, 2001, http://www.nytimes.com/2001/03/08/health/08PARK.html.

[33] C. Warren Olanow et al., “A Double-Blind Controlled Trial of Bilateral Fetal Nigral Transplantation in Parkinson’s Disease,” Annals of Neurology 54, no. 3 (2003): 403–14, https://doi.org/10.1002/ana.10720.

[34] Rebecca. D. Folkerth and Raymon Durso, “Survival and Proliferation of Nonneural Tissues, with Obstruction of Cerebral Ventricles, in a Parkinsonian Patient Treated with Fetal Allografts,” Neurology 46, no. 5 (1996): 1219, https://doi.org/10.1212/wnl.46.5.1219; C. Dirk Keene et al., “A Patient with Huntington’s Disease and Long-Surviving Fetal Neural Transplants That Developed Mass Lesions,” Acta Neuropathologica 117, no. 3 (2009): 329–38, https://doi.org/10.1007/s00401-008-0465-0.

[35] Heiko Braak and Kelly Del Tredici, “Assessing Fetal Nerve Cell Grafts in Parkinson’s Disease,” Nature Medicine 14, no. 5 (2008): 483, https://doi.org/10.1038/nm1768.

[36] Ninette Amariglio et al., “Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient,” PLoS Medicine 6, no. 2 (2009): e1000029, https://doi.org/10.1371/journal.pmed.1000029; “Stem Cell ‘Cure’ Boy Gets Tumour,” BBC News, February 18, 2009, http://news.bbc.co.uk/2/hi/health/7894486.stm.

[37] Harvey Lodish et al., Molecular Cell Biology, 4th ed. (New York: W. H. Freeman, 2000): 183–86.

[38] Leonard Hayflick and Paul S. Moorhead, “The Serial Cultivation of Human Diploid Cell Strains,” Experimental Cell Research 25, no. 3 (1961): 585–621, https://doi.org/10.1016/0014-4827(61)90192-6; F. L. Graham et al., “Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5,” Journal of General Virology 36, no. 1 (1977): 59–72, https://doi.org/10.1099/0022-1317-36-1-59; Bo Ma et al., “Characteristics and Viral Propagation Properties of a New Human Diploid Cell Line, Walvax-2, and Its Suitability as a Candidate Cell Substrate for Vaccine Production,” Human Vaccine Immunotherapeutics 11, no. 4 (2015): 998–1009, https://doi.org/10.1080/21645515.2015.1009811.

[39] Leonard Hayflick, “The Limited In Vitro Lifetime of Human Diploid Cell Strains,” Experimental Cell Research 37, no. 3 (1965): 614–36, https://doi.org/10.1016/0014-4827(65)90211-9; Hayflick and Moorhead, “The Serial Cultivation of Human Diploid Cell Strains.”

[40] Sander Lee et al., “Human Fetal Tissue from Elective Abortions in Research and Medicine.”

[41] Hayflick and Moorhead, “The Serial Cultivation of Human Diploid Cell Strains.”

[42] Leonard Hayflick et al., “Preparation of Poliovirus Vaccines in a Human Fetal Diploid Cell Strain,” American Journal of Hygiene 75, no. 2 (1962): 240–58, https://doi.org/10.1093/oxfordjournals.aje.a120247.

[43] J. P. Jacobs, C. M. Jones, and J. P. Baille, “Characteristics of a Human Diploid Cell Designated MRC-5,” Nature 227, no. 5254 (1970): 168–70, https://doi.org/10.1038/227168a0.

[44] Ma et al., “Characteristics and Viral Propagation Properties of a New Human Diploid Cell Line, Walvax-2.”

[45] W. W. Nichols et al., “Characterization of a New Human Diploid Cell Strain, IMR-90,” Science 196, no. 4285 (1977): 60–63, https://doi.org/10.1126/science.841339.

[46] W. W. Nichols et al., “Characterization of a New Human Diploid Cell Line—IMR-91,” In Vitro 19, no. 10 (1983): 797–804, https://doi.org/10.1007/BF02618099.

[47] Jacobs, Jones, and Baille, “Characteristics of a Human Diploid Cell Designated MRC-5”; Nichols et al., “Characterization of a New Human Diploid Cell Strain, IMR-90,” 60; Nichols et al., “Characterization of a New Human Diploid Cell Line—IMR-91”; Ma et al., “Characteristics and Viral Propagation Properties of a New Human Diploid Cell Line, Walvax-2.”

[48] Graham et al., “Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5.”

[49] Frits J. Fallaux et al., “New Helper Cells and Matched Early Region 1-Deleted Adenovirus Vectors Prevent Generation of Replication-Competent Adenoviruses,” Human Gene Therapy 9, no. 13 (1998): 1909–17, https://doi.org/10.1089/hum.1998.9.13-1909.

[50] Graham et al., “Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5”; F. L. Graham, “Cell Line Transformation,” Current Contents 8, no. 8(1992): 2, http://www.garfield.library.upenn.edu/classics1992/A1992HC31200001.pdf.

[51] U.S. FDA Center for Biologics Evaluation and Research, Vaccines and Related Biological Products Advisory Committee, “Meeting Transcript,” May 16, 2001, https://wayback.archive-it.org/7993/20170404095417/https:/www.fda.gov/ohrms/dockets/ac/01/transcripts/3750t1_01.pdf; Alvin Wong, “The Ethics of HEK 293,” The National Catholic Bioethics Quarterly 6, no. 3 (2006): 473–95, https://doi.org/10.5840/ncbq20066331.

[52] Yao-Cheng Lin et al., “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations,” Nature Communications 5, no. 4767 (2014): https://doi.org/10.1038/ncomms5767.

[53] Graham, “Cell Line Transformation.”

[54] Lin et al. “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations.”

[55] “Cellosaurus HEK293 (CVCL_0045),” Expasy, December 16, 2021, https://web.expasy.org/cellosaurus/CVCL_0045.

[56] U.S. FDA CBER, Vaccines and Related Biological Products Advisory Committee, “Meeting Transcript.”

[57] Y. C. Lin et al. “Genome Dynamics of the Human Embryonic Kidney 293 Lineage in Response to Cell Biology Manipulations,” 4767; Nathalie Louis, Carole Evelegh, and Frank L. Graham, “Cloning and Sequencing of the Cellular–Viral Junctions from the Human Adenovirus Type 5 Transformed 293 Cell Line,” Virology 233, no. 2 (1997): 423–29, https://doi.org/10.1006/viro.1997.8597; L. Bylund et al., “Analysis of the Cytogenetic Stability of the Human Embryonal Kidney Cell Line 293 by Cytogenetic and STR Profiling Approaches,” Cytogenetic and Genome Research 106, no. 1 (2004): 28–32, https://doi.org/10.1159/000078556; A. A. Stepanenko and V. V. Dmitrenko, “HEK293 in Cell Biology and Cancer Research: Phenotype, Karyotype, Tumorigenicity, and Stress-Induced Genome-Phenotype Evolution,” Gene 569, no. 2 (2015): 182–90, https://doi.org/10.1016/j.gene.2015.05.065.

[58] Fallaux et al., “New Helper Cells and Matched Early Region 1-Deleted Adenovirus Vectors Prevent Generation of Replication-Competent Adenoviruses”; U.S. FDA Center for Biologics Evaluation and Research, Vaccines and Related Biological Products Advisory Committee, “Meeting Transcript.”

[59] Tanja Deurholt et al., “Novel Immortalized Human Fetal Liver Cell Line, cBAL111, Has the Potential to Differentiate into Functional Hepatocytes,” BMC Biotechnology 9, no. 89 (2009), https://doi.org/10.1186/1472-6750-9-89.

[60] Philippe Ravassard et al., “A Genetically Engineered Human Pancreatic β Cell Line Exhibiting Glucose-Inducible Insulin Secretion,” The Journal of Clinical Investigation 121, no. 9 (2011): 3589–97, https://doi.org/10.1172/JCI58447.

[61] Kenta Umehara et al., “A New Conditionally Immortalized Human Fetal Brain Pericyte Cell Line: Establishment and Functional Characterization as a Promising Tool for Human Brain Pericyte Studies,” Molecular Neurobiology 55, no. 7 (2018): 5993–6006, https://doi.org/10.1007/s12035-017-0815-9.

[62] Wong, “The Ethics of HEK 293.”

[63] Sander Lee et al., “Human Fetal Tissue from Elective Abortions in Research and Medicine.”

[64] Wong, “The Ethics of HEK 293”; James Bopp Jr., “Fetal Tissue Transplantation and Moral Complicity with Induced Abortion,” in The Fetal Tissue Issue: Medical and Ethical Aspects, ed. Peter Cataldo and Albert Moraczewski (Braintree, MA: Pope John Center, 1994): 61–79.

[65] Also addressed elsewhere in this Special Report.