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Research Papers On Embryonic Stem Cell Research

Summary

Research in human induced pluripotent stem cells (hiPSCs) is rapidly developing and there are expectations that this research may obviate the need to use human embryonic stem cells (hESCs), the ethics of which has been a subject of controversy for more than 15 years. In this study, we investigated approximately 3,400 original research papers that reported an experimental use of these types of human pluripotent stem cells (hPSCs) and were published from 2008 to 2013. We found that research into both cell types was conducted independently and further expanded, accompanied by a growing intersection of both research fields. Moreover, an in-depth analysis of papers that reported the use of both cell types indicates that hESCs are still being used as a “gold standard,” but in a declining proportion of publications. Instead, the expanding research field is diversifying and hESC and hiPSC lines are increasingly being used in more independent research and application areas.

Introduction

With the first reports on generating human induced pluripotent stem cells (hiPSCs) from human cells (Takahashi et al., 2007; Yu et al., 2007), the controversy regarding the ethics of research involving human embryonic stem cells (hESCs) (Thomson et al., 1998) has arisen once again (Holm, 2008). Opponents of hESC research have been quick to argue that, considering the availability of an alternative source of human PSCs (hPSCs), research in hESCs is no longer needed to realize the promise of hPSCs. However, even before the derivation of hiPSCs was first reported, leading scientists in the field of hPSC research emphasized the need to continue research in ESCs in case hiPSCs became available (Hyun et al., 2007).

Several arguments have been put forward to support the continuation or even an extension of hESC research. For example, it has been reasoned that hESCs have advantages over hiPSCs for regenerative therapies because the latter may contain somatic mutations or reprogramming-induced epigenetic defects. Indeed, there are currently 11 clinical trials registered with the FDA in which hESC-derived cells are being used, mainly to establish treatments for different forms of macular degeneration, but also for neurological, cardiac, and pancreatic disorders (NIH, clinicaltrials.gov; https://clinicaltrials.gov/). Although the first results from one of the studies on macular degeneration have been reported (Schwartz et al., 2015), the vast majority of these trials started very recently, at a time when hiPSCs have already been available for years. Currently, hiPSC-derived cells are being used in one clinical trial in Japan (UMIN Clinical Trial Registry, ID UMIN000011929; http://www.umin.ac.jp/ctr/). Another argument in favor of continuing the use of hESCs is their utility for basic research (e.g., to gain a better understanding of human ground-state pluripotency) (Gafni et al., 2013), for studies of early human development (Niakan et al., 2012), or as cells that are unimpeded by epigenetic or environmental disturbances that are likely present in hiPSCs (e.g., to study gene function in a rather naive cell).

One of the most widely used arguments to justify hESC research is that these cells are still needed as the “gold standard” for human pluripotency to characterize and qualify hiPSC lines and gain a deeper understanding of the reprogramming process. This argument is frequently used in the political debate among stem cell researchers and proponents of hESC research, and has become a central point in the attempt to justify continued support for this research, for example, by the European Union. Thinking this argument through implies that research into hESCs would mainly lead to a more complete understanding of induced pluripotency and would become more and more dispensable with increasing progress in hiPSC research. Indeed, although novel and less invasive methods for reprogramming somatic cells to pluripotency have been developed in recent years, and some difficulties in the reprogramming procedure have been overcome (Anokye-Danso et al., 2011; Kim et al., 2009; Warren et al., 2010; Yoshioka et al., 2013; Yu et al., 2009), many controversial studies have reported differences between the two types of hPSCs on both genetic and epigenetic levels (Liang and Zhang, 2013; Ma et al., 2014) that may, for example, result in deviant behaviors in specific differentiation settings (Bar-Nur et al., 2011; Hu et al., 2010; Mills et al., 2013). Thus, it is currently unequivocally crucial to use hESCs as a reference material to gain a deeper understanding of hiPSC biology and to improve reprogramming strategies.

However, at present, the degree to which studies of hESCs and hiPSCs overlap, whether hESCs are being increasingly replaced by hiPSCs, and the purposes for which hESCs are used in iPSC research remain unknown. Six years after the onset of research into hiPSCs, scientific projects that were planned and started after hiPSCs became available should now be completed and published, and a meta-analysis of the relevant papers can be performed to indicate trends with respect to the relationship between hESC and hiPSC research. For example, if hESC research were just a transient technology and hESCs were mainly used as a “gold standard” in hiPSC research, one would expect the extent of independent hESC research to have declined in recent years and the cells to be mainly used in the context of comparative studies with hiPSCs.

In this study, we aimed to address these issues and provide a substantiated and validated database to facilitate further discussion. We analyzed all original research papers involving the experimental use of hPSCs that were published after the onset of hiPSC research. This analysis revealed that although research in hESCs and hiPSCs co-exists, both areas are growing into independent and autonomous research fields that increasingly intersect. About one-quarter of studies involving hESCs were found to also involve hiPSCs. Furthermore, a close inspection of the overlap of hESC and hiPSC studies showed that in the majority of these studies, hESCs were not used as a mere “gold standard” to qualify and better understand hiPSCs, and that this role of hESCs is declining while their use is diversifying and increasing in other areas.

Results

Database Searches and Paper Selection

We performed extensive searches of the PubMed database for studies that reported an experimental use of hESCs or hiPSCs and were published from 2008 to 2013. Our searches resulted in 11,137 hits for hESC-related studies and 6,291 hits for hiPSC-related studies. Of the identified studies, we excluded 3,313 and 2,444 studies, respectively, that were categorized by PubMed as non-original research (e.g., comments, editorials, and reviews). In addition, we excluded 473 hESC and 227 iPSC papers because they appeared in journals that do not publish original experimental research. To identify studies reporting original research in hPSCs, we manually inspected abstracts and/or full texts of the remaining 7,351 hESC and 3,620 hiPSC papers for the use of hESCs and/or hiPSCs, and excluded articles of no relevance for our analysis (e.g., studies reporting on research in mouse or non-human primate stem cells, somatic human stem cells, or political or ethical aspects of research).

This paper-selection procedure finally resulted in a pool of 2,922 studies reporting on experimental use of hESCs (38.4% of the studies inspected manually) and 1,376 studies reporting on experimental use of hiPSCs (36.2% of studies inspected manually). These publications were used for subsequent analyses, and the full texts of these papers were also investigated to identify the specific hESC and/or hiPSC lines used in the studies.

Research Involving hESCs

In the course of our analysis of the 2,922 studies involving experimental use of hESCs, we first identified papers in which hESCs were solely used for comparison with hiPSCs (as a “gold standard,” e.g., to compare novel hiPSC lines with hESCs with respect to pluripotency marker gene expression; see Experimental Procedures for criteria). We identified 401 papers that used hESCs only for comparative reasons and thus provided no inherent contribution to hESC research. Therefore, these papers were excluded from the hESC paper pool for the analyses of hESC research trends. However, these 401 papers were included again for the subsequent analysis of research involving both hESCs and hiPSCs (see below).

The distribution of the remaining 2,521 (not “gold standard”) hESC research papers on a timeline ranging from 2008 through the end of 2013 is shown in Figure 1A. To illustrate long-term trends during the whole period of hESC research, we also included the respective data for the years before 2008 that were derived in a previous study (Löser et al., 2008). Altogether, the number of hESC papers published per year steadily increased throughout the whole era of hESC research, with a minor decline in 2011. Although there apparently has been a slower increase from 2010 on, the result clearly indicates a sustained high interest in hESCs despite the worldwide availability of hiPSCs.

We next investigated the regional distribution of hESC research. By the end of 2013, research groups located in 38 nations reported results of experimental hESC research in international scientific journals. To determine the contribution of individual nations to worldwide hESC research, we allocated each paper to a specific country according to the affiliation of the corresponding author. This approach is justified since about 73% of the hESC research papers published so far resulted from national research with scientists from only one country mentioned in the authors list in the respective paper. The results of our analysis are presented in Figure 1B for the complete era of hESC research (2000–2013) and for the years 2000–2009 and 2010–2013. Our results confirm the unchallenged leadership in hESC research by groups located in the United States, which continuously contributed about 40% of publications to international hESC research for the past one and a half decades. The contribution of United States-based researchers remained nearly unaltered during the two periods shown. This is interesting because these results reflect the research output under the two fundamentally different stem cell policies of the Bush and Obama administrations (2000–2009 and 2010–2013, respectively). In contrast to the United States, the relative contribution to worldwide research from some other nations that entered the hESC field very early, such as Israel, Sweden, and Singapore, markedly declined in the second period, whereas the research output from countries that entered the hESC research field after a delay, such as Germany, France, and Spain, increased to some degree. Notably, the share of research from China (including Hong Kong and Taiwan) in international hESC research increased from less than 5% in the 2000–2009 period to more than 8% in the second period (2010–2013). We also related the number of hESC papers published in the 2008–2013 period to the overall number of publications in the life and health science fields. As shown in Figure S2A, the share of hESC research papers in the overall publication number slightly increased from 2008 to 2013, with some regional differences.

To exclude distortion of this comparison, the number of hESC research papers was related to the overall research output of the respective country in the fields of life and health science (Figure S2A). For example, it became apparent that Singapore, Israel, and Finland overproportionally published research in the hESC field, whereas there was no major imbalance toward this field in other countries.

Research Involving hiPSCs

We next determined the extent of research involving hiPSCs through the end of 2013. As shown in Figure 2A, the number of papers reporting on experimental use of hiPSCs substantially increased from 2007 (the year of the first publication regarding hiPSCs, with only two original research papers in the field) to 2013. We divided the hiPSC era into two periods: 2008–2010 and 2011–2013. While only 267 hiPSC research papers were published in the first 3 years, the number more than quadrupled to 1,109 papers in the following 3-year period. This seems to be a usual phenomenon after the establishment of a novel research field: in the case of hESC research, the output of research even increased more than 8-fold in the second 3-year period of hESC research (2003–2006) compared with the first 3-year period after the first derivation of hESCs (2000–2002). Notably, in 2013 the output from both fields of hPSC research was at a comparable level (about 500 papers each).

By the end of 2013, research groups from 27 nations contributed to research into hiPSCs. As in the case of hESCs, the leadership of US-based researchers is unchallenged, with an overall share of 45% in hiPSC research published worldwide, although the contribution declined from more than 50% in the 2008–2010 period to about 43% in the more recent period (Figure 2B). As expected, when the number of hiPSC research papers was related to the overall publication numbers in life and health sciences from selected countries, we observed a strong increase in the share of hiPSC papers for all countries from 2008 to 2013 (Figure S2B). However, there are some regional differences. Although hiPSC research accounted for less than 0.1% of research from the United States published from 2011 to 2013, it represented more than 0.2% of research from Israel or Singapore in the same period.

Impact of Research in hPSCs

The number of studies originating from an individual country does not necessarily mirror the relevance of that country’s contribution to a research field. We therefore determined, as a measure of the impact of research, the average frequencies with which hESC and hiPSC research papers published from 2008 to 2012 were cited through the end of 2013. Since reliable and comparable citation frequencies are not yet available for papers published in 2013, those papers were not included in the analysis.

As shown in Figure 3A, papers from the hESC research field were cited at an average frequency of 9.1 per year, whereas papers reporting experimental work involving hiPSCs were cited more frequently (19.4 citations per paper and year). In the hESC field, research papers from the United States, The Netherlands, and Canada were cited more often than the average and far more than studies originating from countries such as China, Korea, Sweden, and Israel, confirming our previous data on the high impact of United States-based hESC research (Löser et al., 2012).

Figure 3

Impact of Research Papers Involving hPSCs from Selected Countries

Similarly, we found notable differences in the impact of hiPSC research from different nations (Figure 3B). Research from the US, Spain, the UK, and Canada overperformed with respect to citation frequency, whereas research papers from other countries, such as Australia, China, and Korea, were cited less frequently. The results are also in agreement with our previous study and confirm the surprising finding on an underperformance of Japanese hiPSC research with respect to impact.

It should be noted that the high average citation number of papers from the hiPSC field is mainly due to the high impact of early work in this field, although we did not include pioneering work from the Yamanaka and Thomson groups (Takahashi et al., 2007; Yu et al., 2007) in our citation analysis. While the average citation number per year only moderately decreased for hESC research papers published from 2008 to 2012, it sharply declined for hiPSC papers (from 92.2 citations per year for studies published in 2008 to 8.6 citations per year for studies published in 2012; Figure S3A). To determine whether the observed diversity in citation frequencies among papers from several nations may be due to extremely frequent citations of only a few highly popular studies, we grouped hESC and hiPSC research papers from selected nations according to their average citation frequency per year (Figure S3B). In the case of hESC research, the share of papers cited at a frequency of more than ∼150% of the average (15 citations per year) was more than 20% among studies from the United States, The Netherlands, and Canada, indicating that a rather broad range of influential hESC papers contributed to the high citation frequency of work from these countries. For hiPSC research, the proportion of papers cited at a frequency of more than 150% of the average (30 citations per year) is highest for work from the United States and The Netherlands (but only about 9%), indicating that the high average citation frequency of hiPSC papers may be partially the result of the high impact of an only moderate number of highly influential papers.

Research Involving hESCs and hiPSCs

In public discussions about the tenability of using hESCs despite the availability of hiPSCs, it is frequently reasoned that hESCs are still needed as a gold standard for the verification and qualification of hiPSCs. Accordingly, hESC research was predicted to be a transient technology that would lead to a complete understanding of hiPSC characteristics and would become dispensable with progress in understanding hiPSC biology. Thus, one would expect that (1) hiPSCs should increasingly replace hESCs; (2) consequently, the number of studies involving hESCs (and not hiPSCs) should decrease over time; and (3) the majority of studies involving hESCs should also involve hiPSCs, and hESCs should be used as a reference material (gold standard) for purposes of comparison only.

To test the validity of these hypotheses, we first analyzed the simultaneous use of hESCs and hiPSCs in experimental research. For this purpose, we scrutinized the full texts of all papers that reported an experimental use of hESCs and were published in 2008–2013 for the use of hiPSCs, and also examined the full texts of all papers that reported an experimental use of hiPSCs and were published in the same period for hESC utilization. The results are shown in Figure 4A. Of the more than 3,400 original research papers involving hPSCs that were published in 2008–2013, more than 2,000 involved the use of hESCs (but not hiPSCs) and almost 500 papers involved the use of hiPSCs (but not hESCs). Work reported in 890 research papers (26.1%) was based on both types of hPSCs. As indicated in Figure 4B, the number of papers based on either hiPSCs only or on both hPSC types markedly increased from 2008 to 2013, whereas the number of studies that were based solely on hESCs (but not on hiPSCs) remained stable at a high level.

Notably, the relative share of hiPSC papers that also involved hESCs markedly declined within the period investigated. While nearly all hiPSC studies published in 2008 also involved hESCs (mainly for the purpose of comparison), this value decreased to about 55% in 2013 (Figure 4C), indicating that an increasing portion of hiPSC research is largely independent of hESCs. This may be partially due to the steadily growing number of papers that report on the derivation and use of hiPSCs in the course of establishing disease-specific cell lines, which usually do not involve ESCs. For example, we identified more than 250 original research papers that reported on the generation of at least one disease-specific hiPSC line. Conversely, the portion of hESC studies that also involved hiPSCs increased from less than 5% to more than 40% in the same period.

Altogether, these results indicate that although the two research fields increasingly intersect, they also exist independently. However, while most of the hESC research published in 2008–2013 did not involve hiPSCs, a large portion of even recent hiPSC research still involved hESCs.

We next analyzed the intersection of 890 papers that reported on experimental work in which both cell types were utilized. Assuming that hESCs are increasingly being used as a gold standard for hiPSC work, one would expect hESCs to be used mainly for the purpose of comparison in these studies. We therefore scrutinized these 890 papers for the specific use of hESCs and categorized them into two groups: one containing 401 papers in which hESCs were only used for purposes of comparison (see Experimental Proceduresfor criteria), and one containing 489 studies in which hESCs were rather autonomous research objects. The latter studies aimed to generate novel information about hESCs and hiPSCs, and were usually intended to gain insight into more general characteristics of hPSCs. Other studies that clustered in this second group were primarily performed with hESCs and results were just verified in hiPSCs to generalize the findings for a second type of hPSCs, or hiPSCs were solely used for the purpose of comparison. The results of this analysis with respect to time course are presented in Figure 5. At the onset of hiPSC research, hESCs were mainly used for mere comparison (about 92% of papers involved both cell types and were published in 2008). However, the share of this kind of paper declined to about 35% in 2013. In contrast, studies belonging to the second group formed the majority of such papers in more recent years.

Figure 5

Type of Use of hESCs in Studies Reporting Experimental Application of Both hiPSCs and hESCs

Trends in Research with hPSCs

Our data indicate that hESCs are not mainly used as the gold standard for hiPSC research. We therefore wished to determine what kinds of scientific questions the different hPSC types were used to address. We roughly categorized papers with respect to the specific use of hESCs or hiPSCs. We restricted our analyses to the years 2011–2013 because during this period a sufficient number of hiPSC lines were available for investigating a broad range of scientific questions, whereas at the onset of hiPSC research (2008–2010), the majority of hiPSC papers focused mainly on the development of novel hiPSC lines.

Results of this analysis are shown in Table 1 (upper panels). Most studies involving hESCs were directed toward the analysis of developmental mechanisms in humans and the development and optimization of protocols to obtain pure populations of mature and functional human cells—mainly neural, cardiac, hematopoietic, and endothelial/vascular cells. A large portion of hESC research also aimed to analyze the cells’ specific characteristics, for example, to identify genes expressed specifically in these cells, to describe their genetic and epigenetic features, or to reveal their biochemical and metabolic peculiarities. A major part of the work was concentrated on uncovering the molecular mechanisms of pluripotency in human cells, optimizing culture protocols for hPSCs, and developing novel methods for their reliable characterization.

Table 1

Topics of Research Involving hESCs and hiPSCs and Published from 2011 to 2013

In contrast, the relative majority of work that involved hiPSCs and was published from 2011 to 2013 aimed at the derivation of hiPSC lines from patients with specific diseases. In many studies, these cell lines were used as cell models for human diseases to reveal differentiation defects or functional deficiencies of the differentiated cells. A large portion of the work was focused on optimizing methods for improved reprogramming and identifying human cell types that are accessible for efficient reprogramming. In this context, the identification of molecules and signaling pathways involved in reprogramming was also of great interest. Work to develop and optimize differentiation procedures was frequently performed in conjunction with hESCs. The determination of characteristics specific to hiPSCs (or to hPSCs in general) and the verification of functional characteristics of hiPSC-derived cells in animal models of human diseases were also major topics of hiPSC research. In contrast to hESC research, which largely depended on previously derived cell lines, novel hiPSC lines were derived in more than half of the studies published from 2011 to 2013, indicating that there is a very large (and steadily growing) pool of hiPSC lines in the international research community (Soares et al., 2014).

We also wished to quantify the relative extent to which hESCs and hiPSCs were used to address questions within the same lines of research. Therefore, we determined the relative share of papers in which hESCs, hiPSCs, or both cell types were used in defined research fields, for example, to develop and optimize differentiation protocols or to establish disease models. As shown in Table 1 (lower panel), the vast majority of research that involved hPSCs and sought to uncover developmental and differentiation mechanisms in humans was done with hESCs only. Similarly, work directed toward the optimization of culture and differentiation protocols mainly involved hESCs. In contrast, the field of disease modeling in conjunction with the establishment of disease-specific cell lines was clearly dominated by hiPSCs. hiPSCs and hESCs were used to comparable extents to develop improved methods for the genetic manipulation of hPSCs or testing of hPSC-derived cells in animal models for human diseases.

Usage Pattern of hESC Lines in Comparative Studies

We previously reported that hESC research is dominated by only a few cell lines (Guhr et al., 2006; Löser et al., 2010) and that patterns of hESC line usage can be easily modeled as a cumulative advantage process (Schuldt et al., 2013). Others have proposed a policy-driven model to explain the preferential use of only a few hESC lines (Scott et al., 2009). Thus, we were interested in determining which hESC lines were the most frequently used for comparison with hiPSCs. To that end, we analyzed the 401 papers (“gold standard”) in which hESCs were used solely for the purpose of comparison. We found that 372 of these papers contained information about the specific hESC line(s) used. The results of our analysis are given in Table 2. Notably, in more than half of the papers (57.4%), the hESC H9 line was used for comparison, followed by the H1 line (29.8%), and frequently both lines were used in the same study. Altogether, the five oldest hESC lines (H1, H7, H9, H13, and H14), which were derived at WiCell as early as 1998 (Thomson et al., 1998), were used as the benchmark in about 74% of the studies to assess the integrity and characteristics of hiPSCs. The use especially of cell line H9 was significantly higher in comparative studies than in overall hESC research.

Table 2

Use of hESC Lines for the Sole Purpose of Comparison in hiPSC Research: 2008–2013

Discussion

To identify global trends in the application of hESCs and hiPSCs in research, we established a curated database of published primary research conducted with these cells between 2008 and 2013, and performed a thorough analysis of studies involving only hESCs or only hiPSCs, as well as intersecting research. The results show that both the hESC and hiPSC research fields increased (hiPSC) or remained at a high level (hESC) with respect to impact and quantitative paper output. Research in which both hPSC types were applied in similar proportions included the development and optimization of cultivation and differentiation protocols, and research on animal models to develop cell-based therapies. Interestingly, we identified early segregation trends for the preferential research use of hESCs and hiPSCs in the recent past. For example, trends for the use of mostly hESCs include basic research on cell pluripotency and plasticity, and analysis of (early) developmental mechanisms. hiPSCs, on the other hand, clearly dominate the field of disease modeling, frequently in conjunction with the derivation of novel disease-specific hiPSC lines and the correction of genetic defects in vitro. Other topics of hiPSC research included the provision of cell models for drug development and toxicity testing, although rather surprisingly, a slight relative overweight of hESCs was found in this application field. This finding may have been influenced by our strict inclusion criteria, which only considered studies that directly used hPSCs, and excluded about 80 studies in which only commercially available hPSC-derived cardiomyocytes, hepatocytes, or neural cells were used. We also excluded other secondary studies that used hPSC-derived nucleic acids, proteins, or data. However, a more likely explanation is the relatively short time span of the research used in this analysis (years 2011–2013). Follow-up studies will be required to establish a trend in this specific area, especially in light of the recent establishment of large-scale hiPSC banking projects to meet the anticipated demand in this field (McKernan and Watt, 2013). It is intriguing that about 20% of the studies involving hiPSCs were focused on the establishment of disease-specific human cell lines, and frequently provided for the first time relevant human cell models for poorly understood, rare, and fatal human diseases (Cherry and Daley, 2013; Peitz et al., 2013). Notably, a large number of projects that aim to derive novel disease-specific hiPSC lines are currently registered with the NIH (ClinicalTrials.gov). While hESCs are a valuable resource for generating isogenic variants for specific diseases on a naive background, and therefore are also playing an increasing role in disease modeling, banking projects involving hESCs are mostly directed toward the distribution of highly characterized lines for comparable basic research and prospective clinical applications (Stacey et al., 2013). Moreover, in many cases, hESCs are used to provide a reliable source for differentiated or progenitor human cells such as neurons and cardiomyocytes, which are not readily accessible in other ways.

The trend for increased differentiation in the field is paralleled by a large and increasing proportion of papers in which both cell types are being used. To analyze the validity of the “gold standard” argument that is frequently used to justify the continued use of hESCs in research, we analyzed the intersection of research papers in which both cell types were applied for their specific uses. Although such intersecting research is increasing in absolute numbers, it includes only a minority (about 26%) of all papers involving hPSCs. Moreover, only a portion of these papers used hESCs solely as a gold standard for comparative research. In addition, the overall proportion of hiPSC studies that also use hESCs is steadily declining, likely because the “gold standard” aspect is less relevant if, for example, the research is focused on disease models or on differentiated progeny derived from hiPSCs. In the same period in which the proportion of hiPSC work involving hESCs declined, the number of hESC-only papers did not decline. These findings may indicate that the trend of field diversification and specification is at least partially due to the specific and differential applicability of these two cell types. These findings also show that hESCs are indeed useful as a gold standard and in general for standardization and benchmarking efforts in the field, but that this is not the major justification for their continued high level of use in research. In addition, the use of hESCs for standardization and comparative research is limited to a very small number of cell lines, which are already well characterized and available from established hESC banks. Hence, it appears that the “gold standard” itself is restricted to a small set: most studies used only a single hESC benchmark line (e.g., H9) rather than a larger, representative panel. The lack of generally accepted standard hPSC lines and insufficient knowledge about acceptable phenotypic tolerances may partly explain this restriction to the most commonly used lines and their quasi-standard status (Adewumi et al., 2007; Boulting et al., 2011; Loring and Rao, 2006; Martí et al., 2013; Nestor et al., 2013). The large hiPSC banks that are currently being established may help to define such benchmark standards.

In the controversial field of pluripotent stem cell research, it is vital to argue on the basis of reliable and solid data that best reflect the actual research situation and are carefully validated. However, available data on recent research activities in this field are often based on abstract searches and automatized algorithms, and not on manually verified data bank searches. For example, Pera and Trounson (2013) estimated the number of publications on hESC research to be nearly 2,000 per year for 2010 through 2012, since review articles were included in their data pool (Pera and Trounson, 2013). More strikingly, the recent European Union-funded Stem Cell Report, published in collaboration with Elsevier and Kyoto University (Barfoot et al., 2013), claimed that more than 500 papers on hESCs were published in 2008, and stated that in 2012, researchers from Germany published substantially more papers in the hESC field than groups from Japan, Korea, or Israel. However, a closer inspection of the data set used for this extensive and highly appreciated study revealed that, for example, the German hESC paper pool contained many publications in which hESCs were not used. Abstract statements such as “Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from hESCs or adult brain tissue. Thus, alternative sources are required” resulted in consideration of the respective paper as a contribution to hESC research. Altogether, nearly 50% of the alleged hESC papers from Germany used for this database did not report any research involving hESCs. Moreover, while our study was under review, Alberta et al. (2015) published an analysis assessing the impact of stem cell research funding programs in selected U.S. states. These authors searched the Web of Science for articles that contained the phrase “human embryonic stem cell” in the title, abstract, or key words, and were (co-)produced by authors with an affiliation in the United States. However, our analysis of this data pool (1,544 hESC-related papers from U.S. authors) revealed that more than 15% of the studies identified by Alberta et al. are not relevant because they did not report research involving hESCs. On the other hand, more than 550 relevant papers from the United States that involved hESCs and are present in our database were missed (including the one by Thomson et al. [1998]). The vast reduction of the initially high number of papers obtained through our selection process confirms that it may be essential for the assessment of research activities to initially generate broader publication-based data pools, and to manually validate each included paper. An analysis of papers only on the basis of meta-data provided by a search engine may result in a massive over- or underestimation of research output and may lead to misleading conclusions, which could potentially influence and misdirect political decision-making.

When compared with our previous studies (Guhr et al., 2006; Löser et al., 2008, 2010, 2012), the current analysis revealed some relevant changes in the number and ranking of countries involved in hESC research. In addition, we substantiated that a country’s quantitative output of papers in hPSC research does not necessarily correlate with the impact of this research. For example, our previous surprising finding that Japan is somewhat underperforming in the hiPSC field was confirmed for recent years with respect to impact per study. Moreover, although the number of hPSC research papers from China increased markedly over the past years, research from China clearly underperformed with respect to impact per study in both the hESC and hiPSC fields. However, it may be expected that this situation will change in the near future as Chinese research groups increasingly publish papers in highly influential, ranking international journals.

Our present study was based on a pool of stem cell publications that only included original research papers. It is a well-reasoned assumption that aspects concerning stem cell history, the prospect of using pluripotent stem cells in future therapies, and the ethical and legal aspects of research cannot reflect the extent of research activities in this field, and their inclusion in such an analysis is irrelevant. Our data on the extent of experimental research involving hPSCs show that both hESCs and hiPSCs supply a vital research field that has not yet reached maturity. The emerging trends of differentiation, diversification, and fusion with other research and technological fields indicate that both hESCs and hiPSCs will be essential and independent components of this research area.

Experimental Procedures

Paper pools were established by searches of the PubMed database, which is accessible through the NIH National Library of Medicine (NIH/NLM). Data bank searches were performed separately to identify hESC- and hiPSC-related publications using the search strings described earlier (Guhr et al., 2006; Müller et al., 2010) and modified as indicated in Supplemental Experimental Procedures. The complete procedures used to identify research papers with relevance for our analysis are described in the Supplemental Information and outlined schematically in Figure S1. Briefly, initial searches of the database resulted in about 17,400 hits (11,137 for hESC-related papers and 6,291 for hiPSC-related papers). We excluded articles that were categorized by PubMed as non-research papers, as well as studies that appeared in journals that do not publish original experimental research. Abstracts and/or full texts of the remaining ∼11,000 papers were inspected manually for the use of hESCs or hiPSCs before they were added to the paper repositories. Therefore, our paper pools only contain original research papers in which hESCs and/or hiPSCs were used experimentally.

To identify papers in which hESCs were merely used as a gold standard for iPSCs, we determined whether hESCs were solely used (1) to determine whether newly derived hiPSCs showed typical characteristics of hPSCs (usually with respect to cell morphology, presence of pluripotency-associated gene products, mRNA and miRNA gene-expression patterns, and/or DNA methylation patterns), (2) to verify that protocols developed for culture and differentiation of hiPSCs would also be applicable to hESCs, or (3) to investigate whether molecular characteristics initially identified in hiPSCs could also be found in hESCs. If hESCs were used for only these comparative purposes, the usage was assigned a “gold standard” application. These studies are not considered as original research in hESCs, and consequently the papers were not included in the analyses of hESC research.

Allocation of a paper to a country was done according to the corresponding author’s affiliation. Citation analysis was performed as described previously (Löser et al., 2012) using the Scopus database. Details are given in Supplemental Experimental Procedures.

Categorization of papers into topic groups was performed by manual inspection of abstracts/full texts, since the use of key words assigned by the publisher and provided by the PubMed and Scopus databases turned out to be an unreliable tool for grouping papers into scientific categories.

Author Contributions

S.K. and A.G. collected and assembled data and performed data analysis. A.K. was responsible for conception and design, data analysis and interpretation, and manuscript writing. P.L. was responsible for conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing.

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Abstract

The use of human embryos for research on embryonic stem (ES) cells is currently high on the ethical and political agenda in many countries. Despite the potential benefit of using human ES cells in the treatment of disease, their use remains controversial because of their derivation from early embryos. Here, we address some of the ethical issues surrounding the use of human embryos and human ES cells in the context of state‐of‐the‐art research on the development of stem cell based transplantation therapy.

Key words: cell therapy/cloning/embryos/ethics/stem cells

Introduction

Human embryonic stem cells (hES cells) are currently discussed not only by the biologists by whom they were discovered but also by the medical profession, media, ethicists, governments and politicians. There are several reasons for this. On the one hand, these ‘super cells’ have a major clinical potential in tissue repair, with their proponents believing that they represent the future relief or cure of a wide range of common disabilities; replacement of defective cells in a patient by transplantation of hES cell‐derived equivalents would restore normal function. On the other hand, the use of hES cells is highly controversial because they are derived from human pre‐implantation embryos. To date, most embryos used for the establishment of hES cell lines have been spare embryos from IVF, but the creation of embryos specifically for deriving hES cells is also under discussion. The most controversial variant of this is the transfer of a somatic cell‐nucleus from a patient to an enucleated oocyte (unfertilized egg) in order to produce hES cells genetically identical to that patient for ‘autologous’ transplantation (so‐called ‘therapeutic’ cloning); this may prevent tissue rejection.

The question ‘Can these cells be isolated and used and, if so, under what conditions and restrictions’ is presently high on the political and ethical agenda, with policies and legislation being formulated in many countries to regulate their derivation. The UK has been the first to pass a law governing the use of human embryos for stem cell research. The European Science Foundation has established a committee to make an inventory of the positions taken by governments of countries within Europe on this issue (European Science Foundation, 2001).

In order to discuss the moral aspects of the isolation and use of hES cells, which is the aim of the present article, it is first essential to understand exactly what these cells are, where they come from, their intended applications and to define the ethical questions to be addressed.

What are (embryonic) stem cells?

‘Stem cells’ are primitive cells with the capacity to divide and give rise to more identical stem cells or to specialize and form specific cells of somatic tissues. Broadly speaking, two types of stem cell can be distinguished: embryonic stem (ES) cells which can only be derived from pre‐implantation embryos and have a proven ability to form cells of all tissues of the adult organism (termed ‘pluripotent’), and ‘adult’ stem cells, which are found in a variety of tissues in the fetus and after birth and are, under normal conditions, more specialized (‘multipotent’) with an important function in tissue replacement and repair.

hES cells are derived from the so‐called ‘inner cell mass’ of blastocyst stage embryos that develop in culture within 5 days of fertilization of the oocyte (Thomson et al., 1998; Reubinoff et al., 2000). Although hES cells can form all somatic tissues, they cannot form all of the other ‘extraembryonic’ tissues necessary for complete development, such as the placenta and membranes, so that they cannot give rise to a complete new individual. They are therefore distinct from the ‘totipotent’ fertilized oocyte and blastomere cells deriving from the first cleavage divisions. hES cells are also immortal, expressing high levels of a gene called telomerase, the protein product of which ensures that the telomere ends of the chromosomes are retained at each cell division and the cells do not undergo senescence. The only other cells with proven pluripotency similar to that of ES cells are embryonic germ (EG) cells, which as their name implies, have been derived from ‘primordial germ cells’ that would ultimately form the gametes if the fetus had not been aborted. In humans, hEG cells were first established in culture in 1998, shortly after the first hES cells, from tissue derived from an aborted fetus (Shamblott et al., 1998). Biologically, hEG cells have many properties in common with hES cells (Shamblott et al., 2001).

In the adult individual, a variety of tissues have also been found to harbour stem cell populations. Examples include the brain, skeletal muscle, bone marrow and umbilical cord blood, although the heart, by contrast, contains no stem cells after birth (reviewed in McKay 1997; Fuchs and Segre, 2000; Watt and Hogan, 2000; Weissman et al., 2000; Blau et al., 2001; Spradling et al., 2001). These adult stem cells have generally been regarded as having the capacity to form only the cell types of the organ in which they are found, but recently they have been shown to exhibit an unexpected versatility (Ferrari et al., 1998; Bjornson et al., 1999; Petersen et al., 1999; Pittenger et al., 1999; Brazelton et al., 2000; Clarke et al., 2000; Galli et al., 2000; Lagasse et al., 2000; Mezey et al., 2000; Sanchez‐Ramos et al., 2000; Anderson et al., 2001; Jackson et al., 2001; Orlic et al., 2001). Evidence is strongest in animal experiments, but is increasing in humans, that adult stem cells originating in one germ layer can form a variety of other derivatives of the same germ layer (e.g. bone marrow‐to‐muscle within the mesodermal lineage), as well as transdifferentiate to derivatives of other germ layers (e.g. bone marrow‐to‐brain between the mesodermal and ectodermal lineages). To what extent transdifferentiated cells are immortal or acquire appropriate function in host tissue remains largely to be established but advances in this area are rapid, particularly for multipotent adult progenitor cells (MAPCs) of bone marrow (Reyes and Verfaillie, 2001). Answers to these questions with respect to MAPCs, in particular whether they represent biological equivalents to hES and can likewise be expanded indefinitely whilst retaining their differentiation potential, are currently being addressed (Jiang et al. 2002; Schwartz et al., 2002; Verfaillie, 2002; Zhao et al., 2002). For other adult stem cell types, such as those from brain, skin or intestine (Fuchs and Segre, 2000), this may remain unclear for the immediate future. Although the discussion here concerns hES cells and the use of embryos, the scientific state‐of‐the‐art on other types of stem cell is important in the context of the ‘subsidiarity principle’ (see below).

Potential applications of hES cells and state‐of‐the‐art

In theory, hES cells could be used for many different purposes (Keller and Snodgrass, 1999). Examples in fundamental research on early human development are the causes of early pregnancy loss, aspects of embryonic ageing and the failure of pregnancy in older women (where genetic defects in the oocyte appear to be important). A second category might be toxicology, more specifically research on possible toxic effects of new drugs on early embryonic cells which are often more sensitive than adult cells (drug screening). The most important potential use of hES cells is, however, clinically in transplantation medicine, where they could be used to develop cell replacement therapies. This, according to most researchers in the field represents the real ‘home run’ and it is the ethics of using embryos in this aspect of medicine that will be discussed here. Examples of diseases caused by the loss, or loss of function, of only one or a limited number of cell types and which could benefit from hES cell‐based therapies include diabetes, Parkinson’s disease, stroke, arthritis, multiple sclerosis, heart failure and spinal cord lesions. Although it is known that hES cells are capable of generating neural, cardiac, skeletal muscle, pancreas and liver cells in teratocarcinomas in vivo in immunodeficient mice as well as in tissue culture, it would be an illusion to consider that cell‐therapies will have widespread application in the short term (i.e. within a couple of years). It is unfortunate that sensational treatment in the media, which implied the generation of whole organs from hES cells, initially left this impression so that the more realistic view emerging is already a disappointment to some patient groups. Nonetheless, a proper scientific evaluation of the therapeutic potential is being carried out in countries that allow the isolation and/or use of existing hES cells. The ethical questions here then also include whether the establishment of new hES cell lines can be justified, in the realisation that eventual therapies, based on either hES or adult stem cells are long‐term perspectives.

There are, at least in theory, various sources of hES cells. In most cases to date, these have been spare IVF embryos, although IVF embryos have been specifically created for the purpose of stem cell isolation (Lanzendorf et al., 2001). In one variant of ‘embryo creation’, it has even been reported that normally organized blastocysts develop from chimeras of two morphologically non‐viable embryos (Alikani and Willadsen, 2002). The most revolutionary option would be the creation of embryos specifically for the purpose of isolating stem cells via ‘nuclear transfer’ (‘therapeutic cloning’). This option is purported to be the optimal medical use of hES technology since the nuclear DNA of the cells is derived from a somatic cell of a patient to receive the transplant, reducing the chances of tissue rejection (see Barrientos et al., 1998; 2000). It is of note that the oocyte in this case is not fertilized, but receives maternal and paternal genomes from the donor cell nucleus. Since by some definitions an embryo is the result of fertilization of an oocyte by sperm, there is no absolute consensus that nuclear transfer gives rise to an embryo (see below).

The establishment of embryonic cell lines is becoming increasingly efficient, with up to 50% of spare IVF embryos that develop into blastocysts after thawing at the 8‐cell stage reported to yield cell lines. There are reports of efficiencies much lower than 50%, however, the quality of the donated embryos being an important determinant of success. Growth of the cell lines over extended periods and in some cases under defined conditions (Xu et al., 2001) has also been reported, but the controlled expansion and differentiation to specific cell types is an area where considerable research will be required before cell transplantation becomes clinical practice (for review, see Passier and Mummery, 2003). In addition, research will be required on how to deliver cells to the appropriate site in the patient to ensure that they survive, integrate in the host tissue and adopt appropriate function. These are the current scientific challenges that will have to be overcome before cell therapy becomes clinical practice; the problems are common to both hES and adult stem cells. The efficiency of establishing embryonic stem cell lines from nuclear transfer embryos is currently unknown, but expected to be lower than from IVF embryos.

Ethical exploration

In the following section, the status of hES cells is first considered. The questions of whether it is acceptable to use pre‐implantation embryos as a source of ES cells for research on cell transplantation therapy and if so, whether embryo use should be limited to spare embryos or may also include the creation of embryos via nuclear transfer (‘therapeutic cloning’), are then addressed.

The status of hES cells

What is the ontological status of hES cells? Should they be considered equivalent to embryos or not? Let us first consider the status of the ‘naked’, isolated inner cell mass (ICM; the source for deriving hES cell lines). The ICM is as it were the ‘essence’ of the pre‐implantation embryo, the precursor of the ‘embryo proper’. The isolated ICM, however, no longer has the potential to develop into a fetus and child, as trophoblast cells, necessary for implantation and nourishment of the embryo, and extra‐embryonic endoderm, are absent. It does not necessarily follow, though, that the isolated ICM is no longer an embryo—we suggest that the whole, isolated ICM could best be qualified as a disabled, ‘non‐viable’ embryo (even though it might, at least in theory, be ‘rescued’ by enveloping the ICM with sufficient trophoblast cells).

What, then, is the status of the individual cells from the ICM once isolated, and the embryonic stem cell lines derived from them? Should we consider these cells/cell lines to be non‐viable embryos too? We would argue that when the cells of the ICM begin to spread and grow in culture, the ICM disintegrates and the non‐viable embryo perishes. Some might argue that hES cells are embryos, because, although hES cells in themselves cannot develop into a human being, they might if they were ‘built into’ a cellular background able to make extra‐embryonic tissues necessary for implantation and nutrition of the embryo. At present this is only possible by ‘embryo reconstruction’ in which the ICM of an existing embryo is replaced by ES cells (Nagy et al., 1993). Commentators who, against this background, regard hES cells as equivalent to embryos, apparently take recourse to the opinion that any cell from which a human being could in principle be created, even when high technology (micromanipulation) would be required to achieve this, should be regarded as an embryo. An absurd implication of this ‘inclusive’ definition of an embryo is that one should then also regard all somatic cells as equivalent to embryos—after all, a somatic nucleus may become an embryo after nuclear transplantation in an enucleated oocyte. It is therefore unreasonable to regard hES cells as equivalent to embryos.

Instrumental use of embryos

Research into the development of cell‐replacement therapy requires the instrumental use of pre‐implantation embryos from which hES cells are derived since current technology requires lysis of the trophectoderm and culture of the ICM; the embryo disintegrates and is thus destroyed. As has already been discussed extensively in the embryo‐research debate, considerable differences of opinion exist with regard to the ontological and moral status of the pre‐implantation embryo (Hursthouse, 1987). On one side of the spectrum are the ‘conceptionalist’ view (‘the embryo is a person’) and the ‘strong’ version of the potentiality‐argument (‘because of the potential of the embryo to develop into a person, it ought to be considered as a person’). On the other side of the spectrum we find the view that the embryo (and even the fetus) as a ‘non‐person’ ought not to be attributed any moral status at all. Between these extremes are various intermediates. Here, there is a kind of ‘overlapping consensus’: the embryo has a real, but relatively low moral value. The most important arguments are the moderate version of the potentiality argument (‘the embryo deserves some protection because of its potential to become a person’) and the argument concerning the symbolic value of the embryo (the embryo deserves to be treated with respect because it represents the beginning of human life). Differences of opinion exist on the weight of these arguments (how much protection does the embryo deserve?) and their extent (do they apply to pre‐implantation embryos?). In view of the fact that up to 14 days of development, before the primitive streak develops and three germ layers appear, embryos can split and give rise to twins or two embryos may fuse into one, it may reasonably be argued that at these early stages there is in principle no ontological individuality; this limits the moral value of an embryo.

Pre‐implantation embryos are generally regarded from the ethical point of view as representing a single class, whereas in fact ∼50–60% of these embryos are aneuploid and mostly non‐viable. For non‐viable embryos, the argument of potentiality does not of course apply. Their moral status is thus only based on their symbolic value, which is already low in ‘pre‐individualized’ pre‐implantation embryos. The precise implications of this moral difference for the regulation of the instrumental use of embryos is, however, beyond the scope of the present article.

The view that research with pre‐implantation embryos should be categorically forbidden is based on shaky premises and would be difficult to reconcile with the wide social acceptance of contraceptive intrauterine devices. The dominant view in ethics is that the instrumental use of pre‐implantation embryos, in the light of their relative moral value, can be justified under certain conditions. The international debate focuses on defining these conditions.

Ethics of using surplus IVF embryos as a source of hES cells

Possible objections are connected to the principle of proportionality, the slippery slope argument, and the principle of subsidiarity.

Proportionality

It is generally agreed that research involving embryos should be related to an important goal, sometimes formulated as ‘an important health interest’ (the principle of proportionality). Opinions differ on how this should be interpreted and made operational. In a number of countries, research on pre‐implantation embryos is permitted provided it is related to human reproduction. Internationally, however, such a limitation is being increasingly regarded as too restrictive (De Wert et al., 2002). The isolation of hES cells for research into cell‐replacement therapies operates as a catalyst for this discussion. It is difficult to argue that research into hES cells is disproportional. If embryos may be used for research into the causes or treatment of infertility, then it is inconsistent to reject research into the possible treatment of serious invalidating diseases as being not sufficiently important. The British Nuffield Council on Bioethics (Nuffield Council on Bioethics, 2000) also saw no reason for making a moral distinction between research into diagnostic methods or reproduction and research into potential cell therapies.

Even if one argued that there is a difference between the two types of research, research on cell therapy would, if anything, be more defensible than research on reproduction. One (in our opinion somewhat dubious) argument is to be found in McGee and Caplan (1999); here the suggestion is made that in using embryos for cell therapy, no embryos are actually sacrificed: ‘In the case of embryos already slated to be discarded after IVF, the use of stem cells may actually lend permanence to the embryo. Our point here is that the sacrifice of an early human embryo, whether it involves a human person or not, is not the same as the sacrifice of an adult because life of a 100‐cell embryo is contained in its cells nuclear DNA.’ In other words, the unique characteristic of an embryo is its DNA; by transplanting cells containing this DNA to a new individual, the DNA is preserved and the embryo therefore not sacrificed—a ‘win–win’ situation for both the embryo and cell transplant recipient. The implication is thus that the use of embryos for cell transplantation purposes is ethically preferable to disposing of them or using them in other (‘truly destructive’) types of research. This extreme genetic ‘reductionism’ is highly disputable and not convincing: the fact that embryos are actually sacrificed in research into cell therapy is masked. A second, more convincing, argument, that the instrumental use of embryos is in principle easier to justify for isolation of hES cells than, for example, research directed towards improving IVF, is that it has potentially far wider clinical implications. It therefore, unquestionably meets the proportionality requirement.

Slippery slope

The slippery slope argument can be considered as having two variants, one empirical and the other logical. The empirical version involves a prediction of the future: ‘Acceptance of practice X will inevitably lead to acceptance of (undesirable) practice Y. To prevent Y, X must be banned’. The logical version concerns the presumed logical implications resulting from the moral justification of X: ‘Justification of X automatically implies acceptance of (undesirable) practice Y’. In this context the problem often lies in the lack of precise definition of X: ‘The difficulty in making a conceptual distinction between X and Y that is sharp enough to justify X without at the same time justifying Y, is a reason to disallow X.’ Both versions of the argument play a role in the debate about the isolation of hES cells for research into cell replacement therapy. An example of the logical version is that acceptance of hES cells for the development of stem cell therapy for the treatment of serious disease automatically means there is no argument against acceptance of use, for example, for cosmetic rejuvenation (Nuffield Council on Bioethics, 2000). The main difficulty is, according to these critics, the ‘grey area’ between these two extremes. One answer to this objection is to consider each case individually rather than reject all cases out of hand. One could use the same objection for example against surgery, which can equally be used for serious as well as trivial treatments.

An example of the empirical version of the slippery slope argument is that the use of hES cells for the development of cell therapy would inevitably lead to applications in germ‐line gene therapy and in therapeutic cloning, then ultimately reproductive cloning. This version of the argument is unconvincing too; even if germ line gene therapy and therapeutic cloning would be categorically unacceptable, which is not self‐evident, it does not necessarily follow from this that the use of hES cells for cell‐therapy is unacceptable. The presumed automatism in the empirical version of the slippery slope argument is disputable.

Subsidiarity

A further condition for the instrumental use of embryos is that no suitable alternatives exist that may serve the same goals of the research. This is termed ‘the principle of subsidiarity’. Critics of the use of hES cells claim that at least three such alternatives exist, which have in common that they do not require the instrumental use of embryos: (i) xenotransplantation; (ii) human embryonic germ cells (hEG cells), and (iii) adult stem cells.

The question is not whether these possible alternatives require further research (this is, at least for the latter two, largely undisputed), but whether only these alternatives should be the subject of research. Is a moratorium for isolating hES cells required, or is it preferable to carry out research on the different options, including the use of hES cells, in parallel?

The answer to this question depends on how the principle of subsidiarity ought to be applied. Although the principle of subsidiarity is meant to express concern for the (albeit limited) moral value of the embryo, it is a sign of ethical one‐dimensionality to present every alternative, which does not use embryos, as a priori superior. For the comparative ethical analysis of hES cells from pre‐implantation embryos on the one hand, and the possible alternatives mentioned on the other, a number of relevant aspects should be taken into account. These include: the burdens and/or risks of the different options for the patient and his or her environment; the chance that the alternative options have the same (probably broad) applicability as hES cells from pre‐implantation embryos; and the time‐scale in which clinically useful applications are to be expected.

A basis for initiating a comparative ethical analysis is set out below:

(i) Xenotransplantation is viewed at present as carrying a risk, albeit limited, of cross‐species infections and an accompanying threat to public health. This risk is, at least for the time being, an ethical and safety threshold for clinical trials. Apart from that, the question may be raised from a perspective of animal ethics whether it is reasonable to breed and kill animals in order to produce transplants, when at the same time spare human embryos are available which would otherwise be discarded;

(ii) In principle, the use of hEG cells from primordial germ cells of dead fetuses seems from a moral perspective to be more acceptable than the instrumental use of living pre‐implantation embryos, provided that the decision to abort was not motivated by the use of fetal material for transplantation purposes. To date, however, hEG cells have been difficult to isolate and culture, with only one research group reporting success (Shamblott et al., 1998; 2001). In addition, research in mice suggests abnormal reprogramming of these cells in culture: chimeric mice generated between mouse (m)EG cells and pre‐implantation embryos develop abnormally while chimeras using mouse (m)ES cells develop as normally as non‐chimeric mice (Steghaus‐Kovac, 1999; Surani, 2001). This makes the outcome of eventual clinical application of these cells difficult to predict in terms of health risks for the recipient.

(iii) Analysis of the developmental potential of adult stem cells is a rapidly evolving field of research, particularly in animal model systems. Experiments carried out within the last two years have demonstrated, for example, that bone marrow cells can give rise to nerve cells in mouse brain (Mezey et al., 2000), neural cells from mouse brain can turn into blood and muscle (Bjornson et al., 1999; Galli et al., 2000), and even participate in the development of chimeric mouse embryos up to mid‐gestation (Clarke et al., 2000). Although apparently spectacular in demonstrating that neural stem cells from mice can form most cell types under the appropriate conditions, it is still unclear whether true plasticity in terms of function has been demonstrated or whether the cells simply ‘piggy‐back’ with normal cells during development. Published evidence of ‘plasticity’ in adult human stem cells is more limited, but recent evidence suggests that the MAPCs from bone marrow may represent a breakthrough (Jiang et al., 2002; Schwartz et al., 2002;). They are accessible. Collection is relatively non‐destructive for surrounding tissue compared, for example, with the collection of neural stem cells from adult brain, although their numbers are low: 1 in 108 of these cells exhibit the ability to form populations of nerve, muscle and a number of other cell types and they only become evident after several months of careful culture. Clonal analysis has provided rigorous proof of plasticity: a single haematopoietic stem cell can populate a variety of tissues when injected into lethally irradiated mice (Krause et al., 2001) or into blastocyst stage embryos to generate chimeric embryos (Jiang et al., 2002). Nonetheless, there are potential hazards to using cells that have been cultured for long periods for transplantation and although MAPCs seem to have normal chromosomes, it is important to establish that the pathways governing cell proliferation are unperturbed. This is also true for hES cells. However, the powerful performance of mES cells in restoring function in a rat model for Parkinson’s disease (Kim et al., 2002), has not yet been matched by MAPCs. Bone marrow stem cells have been shown very recently to restore function to some extent in a mouse heart damaged by coronary ligation, an experiment that mimics the conditions of the human heart soon after infarction (Orlic et al., 2001). Although clinical restoration of function in a damaged organ is usually sought rather longer after the original injury than in these experiments, which were performed before scar tissue had formed, this approach will certainly be worth pursuing. An alternative, non‐invasive, haematopoietic stem cell source is umbilical cord blood. This is used clinically for transplantation as an alternative to bone marrow in patients for whom no bone marrow match is available. Cord blood contains precursors of a number of lineages but its pluripotency, or even multipotency, is far from proven. Nevertheless, the prospect of autologous transplantation of haematopoietic stem cells of bone marrow in the long term makes this an important research area in terms of alternatives to therapeutic cloning (see below).

Although studies with adult stem cells so far have been encouraging, Galli (2000), author of the first adult neural stem studies and much cited by advocates of the view that adult stem cells have a proven developmental potency equal to that of ES cells, himself disagrees entirely with this viewpoint (see Editorial, 2000). It has even been suggested that the results from adult stem cell research are being misinterpreted for political motives and ‘hints of the versatility of the adult cells have been over interpreted, overplayed and over hyped’ (Vastag, 2001). Opponents of ES cell research are now heralding Verfaillie’s adult stem cells as proof that work on hES cells is no longer needed. However the stem cell research community and Verfaillie herself (Vastag, 2002) have called for more research on both adult and embryonic stem cells. ES cells that can perform as powerfully as those described by Kim et al. (2002) in the rat Parkinson model make it far too early in the game for them to be discounted (Editorial, 2002).

The question remains, however, should a moratorium be imposed on isolating hES cells for research in cell therapy in the light of the indisputably promising results from adult stem cell research? The lack of consensus arises largely from disagreement on interpretation of the subsidiarity principle. Against the restrictive viewpoint that research on hES cells may only take place if there is proof that adult stem cells are not optimally useful, there is the more permissive viewpoint that hES cell research may, and indeed should, take place so long it is unclear whether adult stem cells are complete or even partial alternatives.

On the basis of the following arguments, a less restrictive interpretation of the subsidiarity principle is morally justified. (Stem Cell Research, 2000) To begin with, the most optimistic expectation is that only in the long run will adult stem cells prove to have equal plasticity and developmental potential as hES cells (and be as broadly applicable in the clinic), and there is a reasonable chance that this will never turn out to be the case. If hES cells from pre‐implantation embryos have more potential clinical applications in the short term, then the risk of a moratorium is that patients will be deprived of benefit. This in itself is a reason to forgo a moratorium—assuming that the health interests of patients overrule the relative moral value of pre‐implantation embryos. Secondly, the simultaneous development of different research strategies is preferable, considering that research on hES cells will probably contribute to speeding up and optimising clinical applications of adult stem cells. In particular, the stimuli to drive cells in particular directions of differentiation may be common to both cell types, while methods of delivery to damaged tissue are as likely to be common as complementary. A moratorium on hES cell research would remove the driving force behind adult stem cell research.

A final variant on adult stem cell sources concerns the use of embryonal carcinoma (EC) cells, a stem cell population found in tumours (teratocarcinomas) of young adult patients. These cells have properties very similar to hES cells. The results of a phase I (safety) trial using these cells in 11 stroke victims in the USA have recently been published and permission granted by the Food and Drug Administration (FDA) for a phase II trial (effectivity) (Kondziolka et al., 2000). The patients received neural cells derived from retinoic acid (vitamin A) treatment of teratocarcinoma stem cells. Although the scientific and ethical consensus is that these trials were premature in terms of potential risk of teratocarcinoma development at the transplant site, all patients survived with no obvious detrimental effects, no tumour formation and in two cases a small improvement in symptoms. After two years, the transplanted cells were still detectable by scanning (Kondziolka et al., 2000). Despite its controversial nature, this trial has nevertheless probably set a precedent for similar trials using neural derivatives of hES, the best controlled differentiation pathway of hES cells at the present time (Reubinoff et al., 2001; Zhang et al., 2001). Proponents believe that such trials would be feasible even in the short term (McKay, 1997). Neural differentiation of hEC cells is fairly easy to induce reproducibly but most other forms of differentiation are not; even if ultimately regarded as ‘safe’, hEC cells will not replace hES cells in terms of developmental potential and are therefore not regarded as an alternative.

In view of both the only relative moral value of pre‐implantation embryos and the uncertainties and risks of the potential alternative sources for the development of cell therapy, a moratorium for isolating human embryonic stem cells is unjustified.

Therapeutic cloning

Before discussing the ethical issues around ‘therapeutic cloning’, the term itself requires consideration. To avoid confusion, it has been proposed that the term ‘cloning’ be reserved for reproductive cloning and that ‘Nuclear transplantation to produce stem cells’ would be better terminology for therapeutic cloning (NAS report, 2002; Vogelstein et al., 2002). Others have pointed out the disadvantage of this alternative term, namely that it masks the fact that an embryo is created for instrumental use. More important in our opinion however, is that the use of the adverb ‘therapeutic’ suggests that hES cell therapy is already a reality: strictu sensu there can only be a question of therapeutic applications once clinical trials have started. In the phase before clinical trials, it is only reasonable to refer to research on nuclear transfer as ‘research cloning’ or ‘nuclear transplantation for fundamental scientific research’, aimed at future applications of therapeutic cloning.

Some consider this technology to be ethically neutral; they claim that the ‘construct’ produced is not a (pre‐implantation) embryo. Qualifications suggested for these constructs include: activated oocyte, ovasome, transnuclear oocyte cell, etc. (Kiessling, 2001; Hansen, 2002) However, to restrict the definition of ‘embryo’ to the product of fertilization in the post‐Dolly era is a misleading anachronism. Although the purpose of therapeutic cloning is not the creation of a new individual and it is unlikely that the viability of the constructed product is equivalent to that of an embryo derived from sexual reproduction, it is not correct to say that an embryo has not been created.

The core of the problem is that here human embryos are created solely for instrumental use. Whether or not this can be morally justified—and if so, under what conditions—has already been an issue of debate for years in the context of the development of ‘assisted reproductive technologies’ (ART). Is it acceptable to create embryos for research, and if so, is therapeutic cloning morally acceptable too?

A preliminary question: is it justified to create embryos for research?

Article 18 of the European Convention on Human Rights and Biomedicine forbids the creation of embryos for all research purposes (Council of Europe, 1996). However, this does not close the ethical and political debates in individual EU member states.