Organ transplantation is a highly utilized treatment for many medical conditions, yet the number of patients waiting for organs far exceeds the number available. The challenges and limitations currently associated with organ transplantation and technological advances in gene editing techniques have led scientists to pursue alternate solutions to the donor organ shortage. Growing human organs in animals and harvesting those organs for transplantation into humans is one such solution. These chimeric animals usually have certain genes necessary for a specific organ’s development inhibited at an early developmental stage, followed by the addition of cultured pluripotent human cells to fill that developmental niche. The result is a chimeric animal that contains human organs which are available for transplant into a patient, circumventing some of the limitations currently involved in donor organ transplantation. In this review, we will discuss both the current scientific and legal landscape of human–animal chimera (HAC) research. We present an overview of the technological advances that allow for the creation of HACs, the patents that currently exist on these methods, as well as current public attitude and understanding that can influence HAC research policy. We complement our scientific and public attitude discussion with a regulatory overview of chimera research at both the national and state level, while also contrasting current U.S. legislation with regulations in other countries. Overall, we provide a comprehensive analysis of the legal and scientific barriers to conducting research on HACs for the generation of transplantable human organs, as well as provide recommendations for the future.

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiation attenuated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiation attenuated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an increase in the number of T-cells, including CD4+ and tissue-resident effector/ effector memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

We report findings from a new survey of US public attitudes toward human-animal chimeric embryo (HACE) research, designed to compare with recently reported Japanese survey data. We find that 59% of the US public can personally accept the process of injecting human induced pluripotent stem cells into genetically modified swine embryos and having human tissues produced in a pig's body transplanted into a human. This is greater acceptance than in Japan, and there is even strong acceptance among those with strong religious affiliations and who self-identify as conservatives. We argue that strong public support for HACE research, as well as the emerging literature suggesting that humanization of research animals is very unlikely, should compel the NIH to lift its current moratorium on HACE research.

Our group previously demonstrated that administration of a CD34-negative fraction of human non- hematopoietic umbilical cord blood stem cells (UCBSC) 48 h after ischemic injury could reduce infarct volume by 50% as well as significantly ameliorate neurological deficits. In the present study, we explored possible mechanisms of action using next generation RNA sequencing to analyze the brain transcriptome profiles in rats with ischemic brain injury following UCBSC therapy. Two days after ischemic injury, rats were treated with UCBSC. Five days after administration, total brain mRNA was then extracted for RNAseq analysis using Illumina Hiseq 2000. We found 275 genes that were significantly differentially expressed after ischemic injury compared with control brains. Following UCBSC treatment, 220 of the 275 differentially expressed genes returned to normal levels. Detailed analysis of these altered transcripts revealed that the vast majority were associated with activation of the immune system following cerebral ischemia which were normalized following UCBSC therapy. Major alterations in gene expression profiles after ischemia include blood-brain-barrier breakdown, cytokine production, and immune cell infiltration. These results suggest that UCBSC protect the brain following ischemic injury by down regulating the aberrant activation of innate and adaptive immune responses.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Blastocyst complementation is an emerging methodology in which human stem cells are transferred into genetically engineered preimplantation animal embryos eventually giving rise to fully developed human tissues and organs within the animal host for use in regenerative medicine. The ethical issues surrounding this method have caused the National Institutes of Health to issue a moratorium on funding for blastocyst complementation citing the potential for human cells to substantially contribute to the brain of the chimeric animal. To address this concern, we performed an in-depth review of the neural transplantation literature to determine how the integration of human cells into the nonhuman neural circuitry has altered the behavior of the host. Despite reports of widespread integration of human cell transplants, our review of 150 transplantation studies found no evidence suggestive of humanization of the animal host, and we thus conclude that, at present, concerns over humanization should not prevent research on blastocyst complementation to continue. We suggest proceeding in a controlled and transparent manner, however, and include recommendations for future research with careful consideration for how human cells may contribute to the animal host nervous system. Stem Cells 2019;37:444-452.

The potential for developing gene therapy technologies is evident from the recent surge in research activity. The use of these emerging technologies, along with blastocyst complementation, could greatly enhance our ability to produce exogenic organs and cells in a relatively short period of time. Furthermore, the generation of human–animal organ chimeras, together with their associated vasculature, could be developed to overcome the global problem of organ shortage for transplantation, as well as complications associated with immune rejection. It will be imperative that we understand the underlying conditions for stem cell growth in a developmental environment, to determine if cross-species chimeras can be therapeutically developed. Importantly, the combination of gene editing and blastocyst complementation has created an entirely new and exciting field of medicine, one that we envision will have a critical role for precision medicine.

Previous studies have demonstrated that the tropism of Zika virus (ZIKV) for human neural stem cells (NSCs) leads to severe brain malformations. The proposed mechanisms underlying these brain malformations are thought to include the induction of apoptosis in neural progenitor/stem cells (Zhang et al., Nucleic Acid Res. 44:8610–8620, 2016). Previously, we observed many similarities between NSCs and brain tumor stem cells (BTSCs) (Wu et al., Stem Cells Dev., 17:173–184, 2008) and sought to determine if BTSC could support ZIKV replication. ZIKV infection of skin cells in vitro is mediated by the receptors AXL, DC-SIGN, TIM1, and TYRO3 (Hamel et al., J Virol. 89:8880–8896, 2015). We examined human embryonic brain tissue and have observed that the putative ZIKV receptor TIM1 is expressed by neuronal and astrocytic progenitor cells. Given the similarities between NSCs and BTSCs we examined human and rodent brain tumor cells for putative ZIKV receptor expression. We found TIM1 expression by human GBM6 glioma cells and murine GL261 gliomas. In tissue culture studies, incubation of ZIKV with human GBM6 cells led to high levels of infection. To determine the effects of ZIKV on tumor growth in vivo, we transplanted GL261 cells into the striatal area of the brain in syngeneic C57B/6 mice. Intra-tumoral injections of ZIKV were administered immediately after intra-cranial tumor implantation. Tumor-bearing mice receiving intra-tumoral injections of ZIKV exhibited prolonged survival in comparison to untreated mice. Together these results suggest that ZIKV may have potential as an oncolytic virus for targeting malignant brain tumors. (This work was supported in part by a grant from the Randy Shaver Cancer Research Foundation).

Background: T cells undergo autoimmunization following spinal cord injury (SCI) and play both protective and destructive roles during the recovery process. T cell-deficient athymic nude (AN) rats exhibit improved functional recovery when compared to immunocompetent Sprague-Dawley (SD) rats following spinal cord transection.Methods: In the present study, we evaluated locomotor recovery in SD and AN rats following moderate spinal cord contusion. To explain variable locomotor outcome, we assessed whole-genome expression using RNA sequencing, in the acute (1 week post-injury) and chronic (8 weeks post-injury) phases of recovery.Results: Athymic nude rats demonstrated greater locomotor function than SD rats only at 1 week post-injury, coinciding with peak T cell infiltration in immunocompetent rats. Genetic markers for T cells and helper T cells were acutely enriched in SD rats, while AN rats expressed genes for T(h)2 cells, cytotoxic T cells, NK cells, mast cells, IL-1a, and IL-6 at higher levels. Acute enrichment of cell death-related genes suggested that SD rats undergo secondary tissue damage from T cells. Additionally, SD rats exhibited increased acute expression of voltage-gated potassium (K-v) channel-related genes. However, AN rats demonstrated greater chronic expression of cell death-associated genes and less expression of axon-related genes. Immunostaining for macrophage markers revealed no T cell-dependent difference in the acute macrophage infiltrate.Conclusions: We put forth a model in which T cells facilitate early tissue damage, demyelination, and Kv channel dysregulation in SD rats following contusion SCI. However, compensatory features of the immune response in AN rats cause delayed tissue death and limit long-term recovery. T cell inhibition combined with other neuroprotective treatment may thus be a promising therapeutic avenue.