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Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
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6

Renal Tissue

There has not been a new treatment for end-stage renal disease (ESRD) developed in nearly 40 years, said Ben Humphreys, the chief of the Division of Nephrology in the Department of Medicine at Washington University School of Medicine in St. Louis. For many patients with kidney disease, the only treatment is dialysis and, potentially, a kidney transplant, but research advances in recent years have generated hope that new therapies based on gene editing, organoids, and even xenotransplantation may one day be available.

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

END-STAGE RENAL DISEASE

Current approaches to addressing ESRD are not optimal, Humphreys said. According to the U.S. Renal Data System annual data report, more than 660,000 Americans are being treated for ESRD, with 468,000 of those patients being treated with dialysis, a procedure that filters a patient’s blood through an “artificial kidney” to remove waste, salt, and extra water from the body; maintain safe levels of potassium, sodium, and bicarbonate in the blood; and control blood pressure (National Kidney Foundation, 2015, 2016a). Dialysis is a life-saving procedure, and without it people with ESRD would die within 2 weeks, Humphreys said. However, it is a costly therapy. ESRD patients typically receive a 4-hour dialysis procedure three times a week, resulting in costs of about $82,000 per year to treat a single patient (U.S. Renal Data System, 2013). While dialysis is a widely accessible treatment and does extend life, patients on dialysis have a much lower life expectancy than their healthy peers. The only alternative treatment to dialysis is kidney transplantation, which costs less in the long term and improves life expectancy, but is less feasible because there are not enough donor kidneys available to meet the needs of a growing number of patients. More than 100,000 ESRD patients are on the transplant list (National Kidney Foundation, 2016), and most will die before they receive a kidney, Humphreys said. The relative success of dialysis, combined with its high costs, has hampered further development in the field by shunting money away from basic research, he said.

The development of stem cell and regenerative medicine approaches to treat kidney disease lags behind the development of some other fields, but there has been remarkable progress in recent years, Humphreys said. Researchers have started using human pluripotent stem (hPS) cells and induced pluripotent stem (iPS) cells to grow kidney organoids (Takasato et al., 2015). The approach involves culturing hPS cells (or iPS cells) and exposing them to a variety of signaling molecules at specific concentrations and times to mimic the conditions of normal in vivo embryonic development. By observing normal embryonic development and analyzing the signaling found in the pluripotent cells that eventually give rise to the kidney, researchers were able to mimic the migration and signaling pathways to culture two unique progenitor cell populations: ureteric bud cells and metanephric mesenchyme cells. These cells are collected, disaggregated, and then recombined via centrifuge to form a pellet composed of both cell types. The cell pellet is cultured over a

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

period of 2 to 3 weeks, during which time the progenitor cells continue to differentiate and self-organize into small, heterogeneous clumps of cells that resemble the basic structure of a kidney. These structures, called organoids, contain roughly 15 different cell types (of a possible 26 cell types) typically found in the mature kidney (Al-Awqati and Oliver, 2002). They also contain nephron-like structures that consist of the proximal tubule, the glomerulus, the distal tubule, and the collecting duct. In the immediate future, these organoids hold promise for several applications, including disease modeling, toxicity testing for drugs, and drug discovery, Humphreys said. While organoids hold the potential to improve the quality and accuracy of research models in the near future, there is also hope that scientists will eventually be able to grow functional kidney tissue intended for clinical applications in vitro using the technique established to develop organoids. There remain significant challenges that must be overcome before this approach may be used in a clinical setting to treat patients; specifically, the technical challenge of scaling up the size and improving the morphology of organoids to more closely match those of a healthy, mature kidney remains a significant hurdle. Currently, the organoids developed in Humphreys’s lab are very expensive to produce and measure only about 5 millimeters, whereas the human kidney is 10–12 centimeters. Organoids developed in vitro also tend to have a “fried egg” morphology because of the effects of gravity, although researchers have addressed this through the use of a miniature bioreactor that can produce a sphere-shaped organoid.

XENOTRANSPLANTATION AND BLASTOCYST COMPLEMENTATION

Alternatives to traditional human kidney transplant, such as the xenotransplantation of pig kidneys into humans and blastocyst complementation, have been explored and continue to remain an attractive opportunity to develop more accessible and functional organs for transplant in ESRD patients, Humphreys said. Pfizer first investigated xenotransplantation of pig kidneys into humans in the mid-1990s, but the research was stopped because of concern over porcine endogenous retroviruses (PERVs), he said. PERV genomes are integrated into the larger genome of a pig and, depending on the class of PERV, can undergo replication in normal pig cells and infect human cells when exposed in culture or via transplant. Unlike other zoonotic pathogens,

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

PERVs cannot be eliminated through traditional approaches such as biosecure breeding.

Numerous studies have proven the infectivity of subclasses PERV-A and PERV-B when primary pig cells are co-cultured with primary human cells in vitro (Le Tissier et al., 1997; Patience et al., 1997), but it is unclear whether the rate of infectivity of an in vivo transplant would result in widespread infection or the development of clinical symptoms (Wilson, 2008). A study conducted in mice with severe combined immunodeficiency found that the transplantation of pig pancreatic islet cells into the mice resulted in limited PERV infections with no related symptoms. Scientists at The Scripps Research Institute have suggested that immunodecificient mice may provide a good model for the study of xenotransplantation in humans, but they cautioned that further research is required (van der Laan et al., 2000). There are few studies of porcine xenotransplantation into humans because of the potential risks associated with PERVs; however, the limited clinical examples of human exposure to pig organs or xenotransplant products have not demonstrated any clinical evidence of PERV infection.

With the advent of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology, there has been renewed interest in the potential of porcine xenotransplantation, because CRISPR/Cas9 may be used to inactivate PERVs in the porcine genome and remove the risk of PERV infection, which previously could not be eliminated. Initial research into this approach was conducted by researchers at Harvard University, who recently reported the successful inactivation of all PERVs found in a porcine kidney epithelial cell line (PK15) using CRISPR/Cas9 technology. Genomic analysis of the PK15 cell line showed 62 copies of PERVs in the cell genome. To accurately target and inactivate all 62 copies, the researchers used polymerase chain reaction to identify distinct, highly conserved DNA sequences unique to PERVs. These sequences, identified as pol genes, code for a reverse transcriptase that is vital for PERV replication and infection. By designing a Cas9 guide RNA to specifically target the pol gene, the researchers were able to achieve a 1,000-fold reduction in in vitro PERV transmission to human cells as compared with non-edited PK15 cells. This approach has only been applied in vitro to the PK15 cell line, but it does demonstrate the possibility for clinical application in the future (Yang et al., 2015). However, even with the use of CRISPR/Cas9 to inactivate PERVs within the porcine genome, xenotransplantation of pig kidneys into humans remains a substantial challenge, Humphreys said, noting that organ

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

rejection remains an issue and that life-long immunosuppression would still be required to maintain tolerance of the transplanted kidney.

Blastocyst complementation, a technique in which a recipient blastocyst is induced to generate exogenic organs resulting in a chimeric organism, has also emerged as a potential approach to growing human kidneys in pigs, Humphreys said. He cited a study published by Nakauchi et al. in 2013, in which the researchers successfully developed apancreatic pigs by introducing transgenes that inhibit pancreatic development into mature oocytes. The procedure resulted in male pancreatogenesis-disabled fetuses that were capable only of developing a vestigial pancreas. The apancreatic pigs were cloned using somatic nuclear cell transfer. Concurrently, the team has induced donor pig embryos to express the protein humanized Kusabira-Orange (huKO), which fluoresces orange. The apancreatic pig embryos were allowed to mature to the morula stage, at which point they were injected with blastomeres from the morula stage donor embryos that expressed huKO. The chimeric host morulae were cultured in vitro and then transferred to the uterus of a recipient sow and allowed to mature until the late-term fetus stage, when they were analyzed for pancreas development. Fetuses from the host blastocysts (non-chimeras) did not develop a pancreas, while those from the chimeric blastocysts and donor blastocysts did develop pancreata. Notably, the pancreata in the chimeric fetuses fluoresced orange, indicating that they were derived from the donor blastomeres. The success of this approach in this study and others provides the basis for research into the production of human organs in pigs. With current CRISPR technology, the potential to create pig embryos that lack kidneys and other target organs is increasingly feasible, Humphreys said, and the injection of human iPS cells into the CRISPR-edited blastocysts could result in the development of pig chimeras that produce human organs for transplant into patients in need. The immunorejection of the xenogenic organ in the host animal and the potential for organs derived of a mix of host animal and xenogenic tissues remain technical challenges (Kemter and Wolf, 2015). While promising, Humphreys said, the approach is still early in the discovery phase, and the scientific technique and complex ethical issues related to the concept will require years of additional research before the technique reaches the clinic (Nagashima and Matsunari, 2016).

There remains tremendous clinical need for new therapies to treat chronic kidney disease, Humphreys concluded. We have not had a new drug in the chronic kidney disease space in decades, he said, noting that

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

the costs of treatment are remarkably high. Kidney organoids are changing the way researchers in the field approach their pursuits, and with continued investment and collaboration, there is reason for cautious optimism, he said.

POLYCYSTIC KIDNEY DISEASE

Polycystic kidney disease (PKD) is a fatal, monogenic disease and is the fourth leading cause of renal replacement therapy, said David Baron, the chief scientific officer of the PKD Foundation. Autosomal dominant PKD (ADPKD), the most common monogenetic kidney disease, is caused by mutations in one of two genes: PKD1, which accounts for about 85 percent of patients with autosomal dominant PKD, and PKD2, which generally results in a more mild phenotype. Spontaneous mutations are responsible for up to 10 percent of patients with ADPKD, with most individual mutations occurring at a low frequency. The correlation between phenotype and genotype is variable, and some mutations are quite rare. There are about 600,000 people in the United States who have been diagnosed with ADPKD, Baron reported (PKD Foundation, 2016). Autosomal recessive polycystic kidney disease is a similar disease that is caused by a mutation in a different gene that is very rare and that affects the kidneys, livers, and lungs of children (National Kidney Foundation, 2016c).

PKD results in the very rapid growth of the kidneys, and the issues that confront patients include hypertension, infection, hematuria, kidney stones, electrolyte imbalance, pain, fatigue, and, ultimately, ESRD and the need for dialysis or transplant. An increased risk of retroperitoneal bleeds is another effect of PKD, Baron said. The long-term impact of retroperitoneal bleeds can be severe since blood transfusions, which can complicate the ability to accept a kidney transplant, are often used to treat them. Because PKD is a systemic ciliopathy, and because most cells of the body contain a primary cilium, the disease affects far more than just the kidneys, Baron said. For example, other manifestations of PKD include mitral valve prolapse, abdominal wall hernias, diverticulosis, and diverticulitis (National Kidney Foundation, 2016c).

ADPKD is a progressive disease, with cysts most likely developing in utero, but patients are frequently not diagnosed until the third or fourth decade of life, Baron said. The kidney grows rapidly over time, and the disease is usually diagnosed when cysts are found by ultrasound,

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

although MRIs and other imaging techniques can show cysts as well. Glomerular filtration rate (GFR) is a surrogate biomarker for PKD, Baron said, but by the time that GFR begins to decline, the number of parenchyma-destroying cysts has grown so substantially that it is unlikely that renal function can be maintained at that point (Grantham et al., 2006). Another way to detect PKD is through genetic testing, which may be appropriate if there is a family history of PKD or if magnetic resonance imaging or ultrasound imaging shows an uncertain diagnosis of PKD (National Kidney Foundation, 2016c). Ideally, therapy for PKD should occur much earlier in the course of the disease, meaning that disease detection must improve and that any therapy will need to be safe and tolerable, potentially for decades, Baron said.

The current treatment approaches for PKD include symptom management through diet and lifestyle and medication, dialysis, and transplantation, although there is a shortage of available kidneys (NIDDK, 2015). There have been advances in transplantation immunology that have improved how people can live and work with transplants. For example, Baron is on a steroid-free regimen. Advances in cardiovascular therapies have resulted in better control of hypertension and of the effects that declining renal function has on the heart. Although there is still controversy over how the mutation actually causes cysts, researchers are continually improving the knowledge base regarding the cellular and molecular mechanisms of the disease, Baron said. However, he noted that there have been no recent therapeutic advances in the United States. A new therapeutic called tolvaptan, a vasopressin V2 receptor antagonist, has been approved in Canada, the European Union, and Japan, but not yet in the United States (Business Wire, 2013; PKD Foundation, 2013). It decreases the growth of cysts over time, but it also causes extreme thirst, polyuria, and an increase in liver enzymes in some patients (ASHSP, 2016). There are a number of ongoing trials for tolvaptan as well as repurposed drugs such as metformin, pioglitazone, niacinamide, tesavatinib, and lanreotide (PKD Foundation, 2013). However, because of the complexity of the disease, it seems unlikely that any one drug will be able to address the multiple pathways of this disease, Baron said. The regulatory path for the approval for new and novel PKD therapeutics is still quite ill-defined, he said.

Investments in regenerative medicine research for PKD have the potential to be hugely cost-effective, Baron said, because the current standard of renal replacement therapy is so expensive. About $4 billion is spent each year on renal replacement therapy for patients with ADPKD,

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

Baron estimated. Funding for renal research needs to be increased, he said, because the “savings are obvious”—the fewer people on dialysis, the greater the benefit to Medicare.

The applications of regenerative medicine to treat PKD are being explored, although potential therapies are still many years away from clinical application. According to Baron, possible regenerative therapies for PKD include

  • embryo selection at the 32-cell stage to avoid the occurrence of PKD;
  • directed drug delivery into the cyst, such as folate receptor targeted delivery of folate-conjugated rapamycin to the cells that line renal cysts in PKD or the use of dimeric immunoglobulin A antibodies to introduce antibodies against growth factors implicated in the development of renal cysts, such as epidermal growth factor, ouabain, TGF-α, TGF-β, TNF-α, and IL-1β (Olsan et al., 2015; Shillingford et al., 2012);
  • autologous genetically “corrected” stem cell infusion;
  • the infusion of exosomes containing corrected forms of the polycystin-1 protein or mRNA; and
  • implants of autologous genetically corrected kidney organoids or non-immunologic hybrid kidneys made from autologous corrected kidney cells seeded onto a non-immunologic bioengineered scaffold.

Most of these potential therapies are years away from the clinic, Baron emphasized, though he noted that because treating PKD involves nephron regeneration or cell repair, advances in this area may be generalizable to other renal diseases or other ciliopathies. Moving forward, well-informed PKD patients will be needed to inform assessments of the risks versus benefits of potential therapies and to assist the Food and Drug Administration and other regulators in deciding what therapies to move forward with, he said.

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

PANEL DISCUSSION

Patient Awareness

Some participants asked if there was any value in promoting awareness and the early diagnosis of PKD, perhaps through kidney volume imaging of children at ages 10 and 20 to see if the size of the kidney has increased. That is possible, Baron said, although there is an ethical dilemma involved with telling a young person that he or she has a disease that has no treatment.

Bioscaffolding and Organoids

The panel was asked about the possibility of using acellular structures or three-dimensional printing with bio-material to create a functioning organ. This is an area of great interest, Humphreys responded, noting that early research indicates that using progenitor cells to “build” a kidney on a scaffold has resulted in some cells differentiating as they should, while others do not. However, this approach is “forcing something that is a little unnatural,” he said. While we should not rule out new ideas, the most promise lies in direct differentiation, when cells “simply do what they want to do” and create nephrons. Right now, he said, it is possible to create about 200 nephrons in a small organoid, but in the future it will be possible to scale up production to produce the million nephrons that are present in an adult kidney. Hoshizaki added that investment in creating kidney organoids may be fruitful not only as a potential therapeutic organ, but as a research and assay tool.

Gene Editing and PKD

Several workshop participants observed that standard genetic editing may not be possible for PKD, because the affected gene is too large for traditional delivery vehicles. Genetic researchers have had success in treating other diseases by targeting second-site suppressors, that is, genes in a second site that prevent or alleviate a disease that would otherwise be present due to a mutation. Because some PKD patients have milder forms of the disease, there was a discussion about whether this could be due to a second-site suppressor and if these genes could be targets for PKD treatment. This idea is worth investigation, Baron said, but many

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×

factors that explain the disease, including environmental factors, are still unknown.

A workshop participant asked about identifying and using targets in the kidney to grow new nephrons in vivo. To date, this approach has not shown success in humans, Humphreys responded. However, the fact that other species—particularly fish—can grow new nephrons indicates that perhaps the developmental signaling pathways could be reactivated in mammals and result in new nephrons. The activation of this pathway would need to be balanced against the potential for carcinogenesis, Humphreys said.

Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 55
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 56
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 57
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 58
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 59
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 60
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 61
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 62
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 63
Suggested Citation:"6 Renal Tissue." National Academies of Sciences, Engineering, and Medicine. 2017. Exploring the State of the Science in the Field of Regenerative Medicine: Challenges of and Opportunities for Cellular Therapies: Proceedings of a Workshop. Washington, DC: The National Academies Press. doi: 10.17226/24671.
×
Page 64
Next: 7 Looking Toward the Future: Concluding Thoughts »
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Regenerative medicine holds the potential to create living, functional cells and tissues that can be used to repair or replace those that have suffered potentially irreparable damage due to disease, age, traumatic injury, or genetic and congenital defects. The field of regenerative medicine is broad and includes research and development components of gene and cell therapies, tissue engineering, and non-biologic constructs. Although regenerative medicine has the potential to improve health and deliver economic benefits, this relatively new field faces challenges to developing policies and procedures to support the development of novel therapies are both safe and effective.

In October 2016, the National Academies of Sciences, Engineering, and Medicine hosted a public workshop with the goal of developing a broad understanding of the opportunities and challenges associated with regenerative medicine cellular therapies and related technologies. Participants explored the state of the science of cell-based regenerative therapies within the larger context of patient care and policy. This publication summarizes the presentations and discussions from the workshop.

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