Ethical and Safety Considerations in Stem Cell-Based Therapy for Parkinson’s Disease

Abstract

Stem cell-based therapy for Parkinson’s Disease (PD) is entering an exciting era with many groups competing to reach the goal of safe and practical clinical application. However, the road to this goal is long and beset by challenging obstacles, among which are Good Manufacturing Practice (GMP) standards, scalability, and regulatory requirements for the final cell product. Of paramount importance is the patient safety of the stem cell-derived dopaminergic neurons, such that each stage of the cell therapy implementation process must be scrutinized for potential safety concerns before introduction to the clinic can be contemplated. In this chapter, we will critically consider the safety regulations and safety strategies of stem cell-based therapy for PD, emphasizing the principal requirements necessary for this new therapeutic approach to benefit PD patients. We will introduce the current safety challenges and the connections between these safety issues and the special characteristics of neural stem cells. In addition, we will summarize the safety standards for stem cell-based therapy currently adopted by leading cell therapy groups and international regulations. Both in vitro and in vivo safety assessment methods will be discussed as they relate to the implementation of these standards. Finally, we will speculate on strategies for further enhancing the safety of stem cell-based therapy for PD.

1. Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease. Characterized by the loss of midbrain dopaminergic neurons (mDANs) in the substantia nigra pars compacta (SNpc), the condition impairs motor function with bradykinesia, tremor, and rigidity being pathognomonic symptoms [1]. Currently, treatment strategies for Parkinson’s Disease, including dopamine replacement therapy and deep brain stimulation, are symptomatic and not curative interventions. They do not stop or even slow the progress of the disease. With the development of technology to create and manipulate different types of stem cells, stem cell-based therapies have become one of the most promising potential remedies for degenerative diseases, among which PD has gained particular attention due to its characteristic predominant loss of a single cell type.

Over the decades since this concept emerged, many efforts have been made to test the feasibility of using cell-based therapy to treat PD. In 1987, for the first time, researchers succeeded in transplanting human fetal midbrain tissue into the brains of PD patients in an open-label trial [2]. The apparent positive results encouraged further development of this approach. In the 2000s, double-blind multi-center clinical trials for fetal transplantation were performed in the US [3, 4]. Though these trials overall did not have positive outcomes, there were subsets of patients with clinical improvement that thus encouraged further research. This process was accelerated with the appearance of stem cell sources including both human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) as a source to replace fetal tissue. For example, an autologous iPSC-derived DA neuron transplantation surgery was performed by investigators at Harvard University in 2019 [5] and that same year, a clinical trial using HLA-matched hiPSC-derived DA neurons to treat PD patients was initiated at Kyoto University Hospital by researchers from the Center for iPS Cell Research and Application (CiRA) [6].

However, not all news about stem cell treatment is encouraging. The relative ease of access or production of stem cells of various types, the popularization of the stem cell idea in the public mind, and the opportunity to profit from the hopes of desperate patients can be a toxic combination. This has resulted in ill-advised and unscrupulous activity that has brought real harm to patients. A famous incident concerns a stroke patient who traveled across several countries to receive stem cell therapy in the hope that this would restore function to his left arm and leg. But in fact, the unlicensed stem cell treatments allowed the stem cells injected into his body to grow into a tumor that left him nearly paralyzed [7]. Also, in 2022, an Italian surgeon was convicted of one count of “causing bodily harm” in a Swedish court for his stem cell-embedded artificial trachea implantation surgery performed on three patients at Karolinska Institute in 2011 and 2012, all of whom died of severe complication [8]. These incidents are reminders that there are some biosafety issues unique to stem cell therapy that are not well publicized and that should receive special attention if the promise of this field is not to be cut short by such incidents.

Also, in 2022, the U.S. Supreme Court overturned Roe v. Wade, reflecting the great controversy over the right to life of embryos. It is difficult to predict where ethical and legal concerns over the capabilities of modern medicine and science will lead. Though initially an appealing alternative to the destruction of human embryos, as they have at least the theoretical ability to develop into full human bodies, stem cells, especially the hESCs, may ultimately also encounter serious ethical issues and cause conflict between people holding different beliefs.

Here we will touch upon some of the prominent ethical and safety issues of stem cell-based therapy for Parkinson’s disease, and introduce some of the measures currently used or proposed to surpass the safety threshold for clinical application.

2. Ethical history of stem cells and cell therapy

With respect to PD specifically, various sources have been applied to acquire dopaminergic cells—adrenal medulla [9], carotid body [10], fetal tissue [11], fetal pig neuronal cells [12], hESCs, hiPSCs, parthenogenetic human ova [13], etc. Different types of stem cell therapy raise different levels of ethical controversy. Here, we introduce the ethical considerations for 3 major active resources for treating PD: fetal tissue, hESCs, and hiPSCs.

2.1 Fetal tissue

Fetal tissue transplantation, the first ever successful proof of the concept in cell therapy, is in a paradoxical sense the least controversial one in an ethical sense. This is because the materials used in fetal transplantation are derived from fetuses that at the point of abortion become nonviable and thus more analogous to a cadaver organ donor.

However, abortion itself is hugely controversial in many societies as already discussed, and the application of fetal transplantation may provide an incentive to increase the rate of intentional induced abortions. The economical or medical benefits behind fetal transplantation technology may thus give rise to a black market for fetal tissue, which may affect pregnant women’s decisions on the fate of their babies in the womb.

Moreover, despite the nonviable status of an aborted fetus, fetal tissue transplantation is under a variety of restrictions that change over different periods. In 2019, US President Trump forbade NIH funding for research projects involving the usage of fetal tissue [14], a restriction that was then overturned 2 years later by the Biden administration [15].

2.2 Human embryonic stem cells

hESCs are derived from the pluripotent inner cell mass of 5- to 7-day-old embryos. The use of these cells for either research or medical applications, which involves the destruction of human embryos, renders this the most ethically controversial approach to stem cell therapy. Therefore, a major debate continues, as it has for decades, about whether to consider human embryos as human life.

The opponents of the application of hESCs, many of whom represent religious viewpoints, believe that human life begins at conception. For example, there is a passage in the Bible saying: “Before I formed you in the womb I knew you, before you were born, I dedicated you, a prophet to the nations I appointed you” (NAB) (Jeremiah 1:5 (calling of Jeremiah narrative)), that illustrates the basis of this thought. If we endow the embryo with all the rights of a human being, then damaging or destroying an embryo to do scientific research is assault or murder.

The proponents, on the other hand, posit that a person is defined by properties such as viable independence from the mother and that this occurs at a much later stage of development than fertilization. Ethical, moral, and religious viewpoints vary on the importance of such factors as self-awareness, further complicating the issue. Even among these proponents, few people believe that the embryo or blastocyst is just a clump of cells that can be used for research without restriction [16], and further issues arise when considering embryos that are otherwise discarded by-products of in vitro fertilization efforts, those produced expressly for the purpose of research or medical use, and those that derive from long term propagated cell lines; in many such scenarios no option for further development exists even if the embryo is not used for medical research.

Such contradictory views make the use of embryonic stem cells much like abortion itself, which is constantly being debated in various settings. Countries around the world have also set different restrictions on the use of embryonic stem cells according to their own religions and customs, and these restrictions are also constantly changing over time, like those for fetal tissue transplantation, due to innumerable cultural and political factors.

For instance, in the US, on August 9, 2001, U.S. President George W. Bush introduced a ban on federal funding for research on newly created hESC lines. Scientists using funding from NIH could only use cell lines created prior to that date. This policy greatly hindered research on hESCs and on stem cell therapy using such lines, since only 21 hESC lines were generally available. Hence, on March 9, 2009, President Barack Obama reversed this policy, giving researchers access to hundreds of new cell lines. However, so long as the Dickey-Wicker Amendment remains in effect, scientists are still unable to create new lines using tax dollars [17]. Moreover, actual enforcement of such laws varies between different states in ways that may also be unpredictable, and the threat of further policy reversals is underscored by the Supreme Court decision mentioned above.

The situation in the European Union is similar to that in the US. Stem cell research using human embryos is permitted in Sweden, Spain, Finland, Belgium, Greece, Britain, Denmark, and the Netherlands; however, it is illegal in Germany, Austria, Ireland, Italy, and Portugal [18].

2.3 Human induced pluripotent stem cells

hiPSCs are derived from somatic cells in mature individuals using Nobel prize-winning technology developed by Yamanaka [19]. Since no embryos are involved, it is possible to avoid many ethical problems haunting hESCs and fetal tissue transplantation.

The major “ethical” issue for hiPSC therapy is its cost. hiPSCs transplantation is more expensive and resource intensive than other forms of cell therapy as well as traditional medical or surgical therapies. How to ensure equitable access in the field of hiPSC treatment and establish a sound insurance system remains a challenge for cell therapy in PD just as it does for CAR-T and other emerging therapeutic innovations.

Even with hiPSCs there is the possibility of opinions holding that hiPSCs, with the ability to differentiate similar to ESCs, should be subject to the same regulations despite their very different origin. Furthermore, with the use of hiPSCs, the human cloning controversy would also enter the mix. Such concerns cannot be discounted as a source of ethical conflicts in the future that must be considered in clinical implementation of PD cell therapy.

3. Safety standards for stem cell-based therapy

As noted above, the core enabling feature of stem cell-based cell therapy, either ESCs or iPSCs, is that the pluripotency of the stem cell enables it to differentiate into virtually any desired cell type, thus potentially providing a stable and sustainable cell resource. With the establishment of hESCs in 1998 [20] and the advent of iPSCs from somatic cells in 2006 [19], hESCs and hiPSCs, with the ability to produce any cell type from all three germ layers, gradually became the major resource for PD cell therapy research by virtue of their pluripotency, availability in theoretically unlimited quantities, and superior logistical and ethical position compared to fetal tissue.

However, this pluripotency is also a source of significant safety concerns compared to fetal tissues. Early phase clinical trials are expressly intended to establish the safety of a novel therapy. But even in clinical trials, the safety of trial subjects must be prioritized, and early phase human studies must be preceded by a rigorous demonstration of safety in preclinical studies, here aimed largely at reducing the risk of generating tumors or uncontrolled proliferation. Ideally, under specific protocols, stem cells will lose their pluripotency and differentiate into specific normal mature cell type, which for PD is the SNpc mature dopaminergic A9 neuron [21]. However, stem cells exist in the tissues of mature animals—they are capable of following pathways other than differentiation and maturation, as well as of following various branches on the tree of terminal differentiation. In practice, cells may remain at various stages, from undifferentiated, to partially, to fully differentiated during the process of differentiation, and this may be affected when the cues and signals of normal embryonic differentiation are not present. This gives rise to safety concerns of potential tumorgenicity. Table 1 illustrates two successive stages along the differentiation pathway from ESCs or iPSCs to dopaminergic neurons, either of which has unwanted proliferative potential: undifferentiated stem cells and a representative partially differentiated cell type—neural stem cells, and summarizes the distinctions between them. The identification and elimination of such cells in vitro is a key component of preclinical safety testing, and can guide the optimization of differentiation protocols.

Table 1.

Differences between undifferentiated cell and neural stem cell.

4. Safety assessment methods for stem cell-based therapy for PD

To create cell therapy products that meet clinical safety criteria, various methods have been used or proposed to regulate cell quality. There has historically been no consensus on methods or thresholds to meet these criteria. To promote the development of stem cell-based cell therapy as a widely available clinical option, the establishment of uniform quality standards and acceptable methods to achieve them will be an important step. ISSCR, The International Society for Stem Cell Research, established in 2002, is a leading organization of professional stem cell scientists all around the world, and its guideline for safety studies is one proposed scheme for doing this [23]. Government regulatory bodies such as the US Food and Drug Administration (FDA) also have established standards that must be met for cell therapy products [24].

In general, cell products must be produced under Good Manufacturing Practice (GMP) conditions, with meticulous recording and complete characterization of standard operating procedures in place. Products must be fully tested for authentic target cell characteristics, microbial contamination, tumorgenicity, biodistribution, toxicology, genetic alterations, safety of ancillary therapeutic components, and in long-term animal model safety studies. In most situations, in vitro and in vivo testing should be combined for both safety and efficacy testing, but animal welfare regulations dictate that animal data should be supplied only where appropriate and informative, and that nonhuman primates should be used only if they are specifically required models [25]. Both male and female animals should be assessed in preclinical safety tests unless there is a scientifically valid reason not to do so.

4.1 Cell characterization

Transplanted cells to be used in clinical trials must first be rigorously characterized to confirm their authenticity, purity, and potential risks. Furthermore, when products fail to meet these specifications, the reasons must be understood and the production process should be refined and improved. Such reasons may include persistent epigenetic memory in iPSC lines leading to anomalous gene expression or off-target gene expression during the differentiation process. Among the characteristics used to confirm the authenticity of cell products are morphology, molecular biology, and cytogenetics.

Morphological characterization generally involves light microscopic examination and comparison to the target cell types. Molecular biological studies are used to define characteristic markers expected to be present on cells and may include analysis of expression or RNA sequencing of single or multiple cell types. Cytogenetics looks specifically at any alterations in the genetic material from karyotype to single-point mutations. Such characterization protocols should be conducted repeatedly at each stage from ESCs or parent cell type for iPSCs, through reprogramming for iPSCs, and at each step of the induced differentiation process, to monitor progress toward the desired final product.

4.2 Toxicity and microbial contamination tests

These tests are conducted on the cells, laboratory processing equipment, and reagents at every stage in accordance with GMP requirements to determine whether the product is contaminated by microorganisms or endotoxins produced by such organisms and to trace their origin if found.

4.3 Tumorgenicity studies

Because of the intrinsic nature of stem cells and the genetic manipulation required for reprogramming, (such risks for tumorigenicity would not apply so strongly to fetal tissue), these risks must be rigorously assessed for any stem cell-based products, as tumorgenicity is the most significant safety concern that needs to be addressed. Moreover, the final product of iPSC-derived differentiated cells themselves may be tumorigenic, as there is a risk due to somatic mutations in the parent cells or later expression of any mutations acquired during reprogramming.

In vitro studies include examining rates of proliferation, with special attention to whether rapidly dividing subclones tend to take over the cultures, and looking for expression of oncogenes or loss of tumor suppressor gene activity. Although these tests may supplement in vivo studies, they cannot substitute for them. In vivo animal experiments often include histological examination of the morphology and composition of cell grafts at various stages (percentage of undifferentiated cells versus the percentage of desired cell product). Positive tumor-generating controls and negative controls are needed. It is necessary to pay attention to differences in timing and developmental patterns between animal and human species and to the special tumor risk factors in immunodeficient animal model systems. The combination of interspecies differences in tumor development between rodents and humans and the immunodeficient status of mice used for xenograft models compromises the translatability of some tumorigenic risks from animals to man. Spiking experiments using the largest feasible animal dose of the therapeutic product mixed with undifferentiated iPSCs in different quantities are usually required.

4.4 Biodistribution studies

Biodistribution studies determine the distribution, persistence, and clearance of a cell therapy product in vivo from the site of injection to target and nontarget tissues and biofluids. For all stem cell-based products, whether injected locally or systemically, researchers should perform detailed and sensitive biodistribution studies of cells to prevent potential risks related to abnormal migration of grafts [26]. Biodistribution studies both within the CNS and between CNS and periphery are important. In this context, the differences between autologous, allogeneic, and immunosuppressed host environments may be important and should be considered.

4.5 Ancillary therapeutic components

Cell-based interventions frequently involve materials other than cells per se, such as biomaterials, engineered scaffolds, and injection devices, as well as the carrier vehicle in which cells are prepared and suspended for transplantation. These materials or surgical instruments must also be tested for safety and effectiveness. Safety and efficacy studies should include an assessment of possible interactions between the cell products and these materials, in vitro and in vivo.

4.6 Long-term safety studies

Long-term safety studies are done after the transplantation surgery is conducted in animals. Given the intended persistence of cells and the irreversibility of some cell-based interventions, testing of the long-term fate and effect of transplanted cells in animal models is vital. As non-human animal models may not replicate the full range of human toxicities associated with stem cell-based interventions, particular care must be applied in preclinical analysis. Health conditions and mortality rates of treated animals should be carefully recorded in an unbiased fashion so that sensitive detection of unexpected adverse effects can be achieved. Beyond this, toxicities and other emerging side effects that are likely to be unique to stem cells or their progeny can also be observed.

4.7 Application of genetic alteration and genome editing technologies to stem cell products

Genetic alteration or genome editing technologies can be coupled to stem cell therapies or applied directly in vivo to resident tissue cells for a variety of therapeutic purposes. If gene editing technology and stem cell-based therapy are combined, researchers should comprehensively investigate the type and genomic distribution of introduced genetic alterations as well as their potential adverse effects on the genome and the biological properties of the treated cells at short and long-term time points. Special attention must be given to the effects of off-target changes in the genome and to the distribution of genetic alterations to unintended cell types in grafts.

5. Methods and technologies in stem cell-based therapy for PD

In stem cell-based therapy for Parkinson’s disease, a variety of approaches have been applied to test the safety of cell products. Here, we provide examples of such approaches and some of the release criteria used by three representative teams that have obtained regulatory approval for PD cell therapy trials, including the laboratories of Kim [5], Studer [27], and Takahashi [28] (Table 2).

Cell type	Biological marker	Release criteria (Lab)
		Kim KS	Studer L	Takahashi J
hiPSCs/hESCs	OCT4	>90%	≥90%	N/A
	SSEA4	>90%	≥90%	N/A
	Nanog	N/A	≥90%	N/A
	TRA-1-60 & TRA-1-81	N/A	≥90%	N/A
DAPs	TRA-1-60	N/A	N/A	<1% (presort)
	CORIN	N/A	N/A	>10% (presort); >90% (postsort)
	FOXA2	>55%; >500-fold than D0	>85%	>80%
	LMX1A	>55%; >500-fold than D0	N/A	N/A
	FOXA2 & LMX1A	>50%	N/A	N/A
	TUJ1	N/A	N/A	>80%
	TH	>10%; >500-fold than D0	N/A	N/A
	OCT3	N/A	<0.1%	<0.1%
	OCT4	None detected	<0.1%	<0.1%
	SSEA4	None detected	N/A	N/A
	TRA-2-49/6E	N/A	N/A	<0.1%
	SOX1	N/A	N/A	<0.1%
	PAX6	N/A	<5%	<0.1%
	Nanog	N/A	<0.2%	N/A
	POU5F1	N/A	N/A	<1% compared to undifferentiated cells
	LIN28	N/A	N/A	<1% compared to undifferentiated cells
	TPH2	<1%	N/A	N/A
	5-HT	<1%	N/A	N/A
	DBH2	<1%	N/A	N/A

Table 2.

An overview of cell characterization release criteria.

N/A: not available.

Method: red: immunostaining; green: flow cytometry; blue: qRT-PCR.

5.1 Cell characterization and counting

The composition and purity of a cell product is perhaps the most important characteristic to be measured. Differentiation protocols applied to either ESC or iPSC do not generally result in monotypic cell cultures, but rather in a mix of cell types related to the embryology of the target region [29]. Immunostaining, flow cytometry, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) are three commonly used methods to count cells by identifying their specific genes or downstream proteins. Each method has its advantages:

• Immunostaining includes immunohistochemistry (IHC), immunocytochemistry (ICC), and immunofluorescence (IF). In vivo immunostaining in particular enables researchers to observe cells without damaging brain structure, thus providing more information about the relationship between graft and host.

• Flow cytometry, compared to immunostaining, is faster and more quantitative and is more often used for in vitro cell counting.

• PCR is a method widely used to rapidly make millions to billions of copies of a specific DNA or RNA samples, and qRT-PCR is a PCR technique used to count the amount of sample, which enables researchers to detect contrasts between samples and note specific cell type signatures.

In developing strategies for stem cell-based therapy for Parkinson’s disease, biological markers for undifferentiated cells and for the end therapeutic product dopaminergic cells (dopaminergic progenitors or dopaminergic neurons) are always measured. Among such markers, OCT4 and SSEA4 are representative markers for undifferentiated cells, FOXA2, and LMX1A are representative markers for dopaminergic progenitors, and TH, TUJ1 are representative markers for dopaminergic neuronal maturity.

A list of marker-based safety criteria from three major laboratories active in the field of PD cell therapy is shown in Table 2. A combined evaluation of biological markers with immunostaining, flow cytometry, and q-RT PCR can provide more detailed information about the composition of the culture.

5.2 Viability

Viability is a significant issue in the production process and during the transplantation of the final products. Many cells are lost during culture for reasons ranging from mechanical disruption during media changes to normal developmental apoptosis. In particular, the viability of the final cell suspension used for transplantation is a critical issue for determining how many cells are actually placed, i.e., the dose, Trypan Blue staining (criteria>70% by Kim Lab), flow cytometry (criteria>90% by Takahashi Lab), and AO/PI staining (criteria≥70% by Studer Lab) have been used for such viability testing.

5.3 Tumorgenicity, biodistribution, and toxicity studies

5.3.1 Tumorgenicity

Although cell characterization can predict the tumorgenicity of cell products through biological markers to some degree, an independent teratoma formation study is indispensable for a thorough tumorgenicity assessment. Such studies require both positive and negative controls. In positive control teratoma formation studies, the parent stem cells (ESCs or iPSCs) are usually injected into the peritoneal cavity or testes of mice to confirm their capability of forming teratomas, and similar analysis may be done with injection at the intended therapeutic target site in the brain. Here, vehicle-only injection serves as the negative control. The final proposed therapeutic product should not result in any tumor formation at the target site. For mice, a sufficient period of time (generally 6–9 months) is allowed to look for more indolent tumor formation. The number of animals used is determined by statistical consideration of the desired threshold for tumor risk which in general should be lower than the background incidence of spontaneous tumor development (Table 3).

Group	Host, administration route	Duration	Number of animals
Tumorigenicity
Takahashi J	NOG, unlesioned, Striatum; NOG, unlesioned, Subcutaneous with Matrigel	12 months; 6 months	N = 80 (DAPs) N = 50 (Saline); N = 20 (DAPs) N = 10 (100% iPSCs) N = 50 spiked with 10% ∼ 0.001% iPSCs
Studer L	NSG, unlesioned, Striatum	9 months	N = 44 (DAPs) N = 44 (with 0.01% & 0.1% hESC) N = 24 (hESCs); N = 24 (vehicle) Male and female
Kim KS	NSG, unlesioned, Striatum	9 months	N = 23 (DAPs)
Biodistribution
Takahashi J	NOG, unlesioned, Striatum	12 months	N = 80 (DAPs) N = 50 (Saline)
Kim KS	NSG, unlesioned, Striatum	9 months	Not mentioned
Studer L	NSG, unlesioned, Striatum	1 month/6 months	N = 10 (High/low dose); N = 10 (Vehicle) Male and female
Toxicology
Takahashi J	NOG, unlesioned, Striatum	12 months	N = 80 (DAPs) N = 50 (Saline)
Studer L	NSG, unlesioned, Striatum	1 month/6 months	N = 20 (High/low dose) N = 20 (Vehicle) Male and female

Table 3.

Summary of preclinical in vivo safety studies.

5.3.2 Biodistribution

Biodistribution studies track the anatomical fate of therapeutic cell products after they are transplanted at the desired dose into the intended location in animal models. As the products are of human origin, they can be distinguished from the host using qPCR or other sensitive techniques. Tissues from different organs or, for PD cell therapy, from multiple brain regions, are collected and assessed for the presence of human genetic material. In PD cell therapy, cell products should be confined to the injection site in the brain or in a particular circuit from the substantia nigra to the striatum, but should not appear inappropriately in other organs or other regions in the brain (Table 3).

5.3.3 Toxicity studies

Phase I clinical trials usually include a dose escalation component designed to look for dose limitations related to toleration of side effects. This is obviously not a practical approach in cell therapy trials where the product is irretrievably implanted into the brain, and thus animal toxicity studies are critical. It is important to bear in mind that extrapolation to humans of certain forms of toxicity such as subtle effects on cognitive function may not be readily assessed in animal models so that conservative interpretations are used. Toxicity studies in animals, therefore, involve transplantation of doses of cell products into the target location, with comprehensive long-term analysis of the health of the animals, recording local and systemic physical and behavioral side effects and cause of death. Wide latitude is applied in drawing conclusions as to whether pathology and cause of death may be related to the cell products or transplantation procedure, with attention again paid to the autologous, allogeneic, or immunosuppressed state of the host (Table 3).

5.4 Contamination

Contamination tests include sensitive detection of viral, bacterial, and mycoplasma presence in the final product or in any of the reagents used to produce it, and may utilize PCR, microbial detection systems for sterility, gram staining for bacteria, endotoxin tests, and adventitious virus testing (Table 4).

Target	Method	Release criteria (Lab)
		Kim KS	Takahashi J	Studer L
Mycoplasma	PCR	Negative	Negative	Negative
Sterility test	BacT/Alert® system	No organism Detected	No organism Detected	No organism Detected
Bacteria	Gram staining	Negative	Negative	Negative
Endotoxin	LAL®	<0.2EU/kg body weight/hr	≤10 EU/mL	≤1 EU/mL

Table 4.

Criteria for contamination test.

5.5 Karyotype analysis and genetic analysis

Karyotype analysis methods include G-band analysis and standard metaphase chromosome analysis, and genetic analysis methods include DNA fingerprinting, whole-genome sequencing, whole-exome sequencing, array comparative genomic hybridization (aCGH), and single-nucleotide polymorphism array (SNP array). Such careful analyses are required to test genomic integrity and to uncover genetic changes, chromosomal aberrations, and karyotype changes in cell products that may relate both to tumorigenicity and to residual epigenetic memory in the final product or aberrant off-target effects of the reprogramming and differentiation process.

6. Strategies to enhance the safety of stem cell-based therapy for PD

6.1 Removal of unwanted cell types

To minimize the potential risks of stem cell therapy for PD, the transplanted products should differentiate to sufficiently pure ventral midbrain dopaminergic (vmDA) neurons, or at a minimum into tissue closely resembling the normal cellular composition of SNpc. To reach this goal, the first step is to remove unwanted cells from the samples.

A major potential risk of stem cell therapy is the possibility that the clinical product may still contain stem cells that are not fully differentiated. These cells have a strong capacity to divide and expand and have a high probability of developing into potentially aggressive tumors. Therefore, before further sorting of the induced mDAPs, candidate products must be treated with inhibitors of cell stemness genes such as c-Myc for the purpose of reducing the number of undifferentiated stem cells.

At the same time, however, care must be taken that the inhibitor system used is not toxic to mDAPs to ensure the survival and growth of mDAPs after transplantation. A good example is the use of the flavonoid quercetin (3,3′,4′,5,7-pentahydroxyflavone), an inhibitor of the gene BIRC5, which shows 99.99% efficiency in removing the undifferentiated hiPSCs but leaves the mDAPs intact [22].

6.2 Target cells purification

Simply removing undifferentiated cells is not sufficient to create an efficacious therapeutic product. mDAPs with specific molecular profiles indicating their commitment to vmDA neuronal fate should be further identified and purified as a second step.

The major techniques currently used to purify target cells from samples can be divided into two main categories:

• Bulk cell sorting methods like filtration, centrifugation, selective cultivation, and magnetic cell sorting (MCS);

• Single-cell sorting methods like fluorescence-activated cytometry sorting (FACS).

In proposed protocols for stem cell therapy, researchers use specific sequences of transcription factors and other manipulations to selectively cultivate a population enriched in the desired target cells. Then either MCS or SACS methods may be applied to further purify the products:

• FACS: This is essentially an extended version of flow cytometry, in which a nozzle creates droplets containing single cells, electrodes apply a charge to those droplets containing cells with target markers, and charged plates create electric fields in order to change the falling trajectory of the droplets so that they sort into different collecting tubes. In FACS, antibodies carrying fluorescent markers bind to specific cell surface markers to label the cells.

• MCS: This method uses magnetic beads encapsulated with antibodies that bind specifically to the target cell surface markers. The target cells with specifically bound magnetic beads are then placed in a magnetic field to separate them out of the suspension. The remaining solutions would be removed, and targeted cells are resuspended for collection.

Table 5 presents a comparison of the two methods:

Criteria methods	Price	Time cost	Versatility	Accuracy
MCS	Lower	Lower	Lower	Lower
FACS	Higher	Higher	Higher	Higher

Table 5.

The comparison between MCS and FACS.

Because FACS involves less manipulation of the cells and is somewhat more versatile in most respects, most published protocols using surface marker-based sorting have employed FACS to purify the cells.

The basis of sorting for both these methods is the diversity of cell surface markers and the presumed presence of specific markers or specific combinations of such markers at critical stages of differentiation to uniquely identify the desired cell types. Therefore, they depend on the ability to find such unique markers and design the corresponding antibodies for FACS. For the cell type ideally suited to our clinical requirements, e.g., dopaminergic neuronal progenitors, the goal is to search for molecular characteristics distinguishing these from other cells, with attention to unique surface markers. This may be accomplished by using single-cell RNA sequencing to look for genes specifically expressed in target cells and then to look for potential surface markers among these genes.

7. Conclusions

Despite decades of recognition of the potential benefits of cell replacement therapy for Parkinson’s disease, progress in this area has been slow. This is related to both ethical and logistical issues in using human fetuses as a tissue source, and to underestimation of the complexities of the disease itself and of the ability to use embryonic tissue to restore function in the adult allogeneic brain. Nonetheless, enough evidence of the potential power of this approach emerged from these efforts that the advent of human ESC and in particular iPSC technology encouraged a renewed enthusiasm and reappraisal of the field that is currently underway. In this chapter, we have discussed some of the differences between the use of fetal tissue that was the proof of principle for these efforts and the use of human ESC or iPSC; the differences between ESC and iPSC; the shared and different risks and goals in using each; and a variety of approaches in use as of this writing to attempt to realize their potential benefits safely and efficaciously. We are optimistic that future editions of this chapter will be able to report significant continued progress toward this important goal.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32170807), and the Natural Science Foundation of Shanghai (21ZR1406300). We also thank members of the Parkinson’s Disease and Cell therapy Laboratory for their discussion and suggestions.

Conflict of interest

The authors declare no conflict of interest.