Summary

Introduction

Since the first derivation of human embryonic stem cells (hESCs) in 1998 (Thomson et al., 1998), research has focused on a better understanding of the unique characteristics of these cells and on a future use of hESC derivatives for cell replacement therapies. However, derivation and use of hESCs has been controversial due to the origin of these cells from human extracorporeal embryos, which are usually destroyed in the process of hESC derivation. With the first establishment of human induced pluripotent stem cells (hiPSCs) in 2007 (Takahashi et al., 2007, Yu et al., 2007) the debate has been raised again. hiPSCs share fundamental characteristics of hESCs but are not burdened with the embryonic origin of hESCs. Since hiPSCs are derived from somatic cells, they can be easily used to establish any disease-specific cell line for analysis of cellular processes during pathogenesis as well as for development of novel agents and drugs. Moreover, because of the option to produce hiPSCs for any diseased individual, they are expected to have advantages over hESCs in future cell and tissue replacement therapies with respect to immune rejection. Some initial disadvantages of hiPSCs, such as dependence on use of retroviruses for reprogramming, have meanwhile been overcome, while others, such as high variability in differentiation potential and genetic stability, still remain subjects of intense research. However, faced with an alternative source for human pluripotent stem cells (hPSCs), it was widely discussed whether hiPSCs would replace hESCs in research and whether hESCs would be exclusively needed to verify the pluripotency of hiPSCs only for a transition period (Holm, 2008, Hyun et al., 2007, Power and Rasko, 2011, Sipp, 2009).

We and others have studied research trends in the field of human pluripotent stem cells over the past 12 years (Guhr et al., 2006, Löser et al., 2010, Negoro et al., 2017, Owen-Smith and McCormick, 2006). Recently, we reported that research with hESCs did not decline but rather broadened from 2008 to 2013 despite the availability of hiPSCs (Kobold et al., 2015). Moreover, we observed a diversification of the research fields in which hESCs and hiPSCs were used to answer partially different research questions. In the current report, we extended this analysis to cover more recent developments. By evaluating all original research papers, which report results of experimental use of hPSCs, we show that there is a considerable increase in hiPSC research while research with hESCs grows only slightly. Furthermore, there is an only limited overlap of both research fields. We also present data on hESC line usage in experimental research and found that the increase in hESC research over the past 10 years is not linked to a considerable rise in the derivation of novel hESC lines. We also present an overview on current clinical trials involving hPSC-derived cell products and investigate the cell line usage in these trials.

Results

Number and Origin of hPSC Research Papers

We first intended to determine the number and origin of research papers that involved experimental use of hESCs and hiPSCs. Figure 1A shows the results of the respective analysis for the past 3 years (2014–2016). In 2014, the number of research papers reporting on hiPSCs equaled the number of hESC studies for the first time. In the following 2 years, the hiPSC paper count clearly surpassed that of hESC papers. Whereas the number of research papers involving hESCs raised only slightly by about 7% from 2014 to 2016, the count of published hiPSC studies increased by more than 55% during the same period. When these numbers were compared to those of the preceding 3-year periods (2008–2010 and 2011 to 2013, respectively), an increase in the number of both hESC and hiPSC research papers by 51.0% and 70.9%, respectively, was noted (Figure 1B). The strong increase in hiPSC research papers clearly indicates the enormous interest in these cells but is also a consequence of the relative novelty of this research field, in which only few publications were produced from 2008 to 2010. We also noted that there is a considerable number of studies in which both hESCs and hiPSCs were used (light gray and gray/white striped portions of the bars in Figure 1B). Closer inspection of stem cell usage in these papers revealed that the number of papers in which hESCs were used for mere comparison to verify certain characteristics of hiPSCs (“"gold standard” usage, gray/white striped portions of the bars in Figure 1B) markedly decreased during the last years indicating that, despite the growth of the hiPSC field, hESCs are still an independent research object. In addition, nine of the hESC studies identified here also report on derivation/experimental use of pluripotent stem cells derived from embryos produced by somatic cell nuclear transfer.

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Figure 1

Worldwide Research in Human Pluripotent Stem Cells

(A) Number of original research papers published from 2014 to 2016. Included are all studies in which experimental use of hESCs (left) or/and hiPSCs (right) is reported.

(B) Number of research papers published on experimental use of hESCs (left) or/and hiPSCs (right) in the 3-year periods indicated. The number of papers in which both hESCs and hiPSCs were used in the same study is shown in lighter gray. Mere gold standard usage of hESCs is indicated by gray-white striped lines.

We next wished to examine the origin of research papers in the hPSC field. About 70% of papers in both the hESC and hiPSC fields were authored by scientists from only one country, while in about 30% authors from laboratories based in at least two countries contributed to the publication. As in our earlier studies, we assigned the latter papers to the country of the corresponding author and excluded papers from the hESC paper pool where hESCs were used for mere comparison with hiPSCs (“gold standard” use of hESCs; for example, when novel hiPSC lines were generated and investigated for their pluripotency in comparison with hESCs, 113 papers for the 2014 to 2016 period). From 2014 to 2016, research groups from 40 nations published results of hESC research, while hiPSC research papers came from groups based in 37 countries. Most papers published in both research fields came from US-based groups, followed by groups from China, the UK, Japan, and Korea (hESC field) and by Japan, China, Germany, and the UK (hiPSC field), respectively. To reveal longer-term trends in the contribution of groups from specific nations to hPSC research, we combined our new data for the 2014 to 2016 period with data collected for the years 2008 to 2013 (Kobold et al., 2015) (Figure 2). This analysis confirmed that most of the 4,744 hESC research papers published during the whole period came from research groups based in the United States (40.5% of all research papers worldwide), followed by groups from China (9.2%), the United Kingdom (7.6%), Japan (4.7%), and South Korea (4.1%, Figure 2A). It should be noted that the US contribution to worldwide hESC research decreased by 15.8% (from 44.3% in the 2008 to 2010 period to 37.3% in the 2014 to 2016 period), whereas the contribution of scientific groups from China increased by 58.6% in the same period (from less than 7%–11.1%). With respect to the 3,544 hiPSC papers published during the whole period, we also noted a sustained leading position of US-based research, which contributed with 41.9% to worldwide published hiPSC research, followed by Japan (14.6%), China (8.3%), Germany (6.3%), and the United Kingdom (4.4%, Figure 2B). As in the hESC field, a relative decrease by nearly 30% in the contribution of US research groups was noted (from 53% in the 2008 to 2010 period to about 37.3% in the 2014 to 2016 period), whereas the relative contribution of groups from China and Germany markedly increased.

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Figure 2

Allocation of hPSC Research to Specific Countries

Share (percentage) of papers from a given country in relation to the total number of hESC research papers (A) and hiPSC research papers (B) for the indicated periods. Only results for countries with more than 50 or 30 original publications in the hESC or hiPSC field, respectively, were included. The numbers to the right of the upper bars relate to the total 9-year period (2008–2016).

Application of hESC Lines in Research

Over the past decade, we and others reported the predominant use of only a few hESC lines in experimental research, which were derived early in the field (Guhr et al., 2006, Löser et al., 2010, McCormick et al., 2009, Scott et al., 2009). However, while some supposed that this phenomenon was caused by a long-lasting and trans-national effect of the research policy of the Bush administration (Scott et al., 2009), we demonstrated more recently that it can be better explained on the basis of power laws (Schuldt et al., 2013). To elucidate whether there are novel trends in the application of certain established hESCs, we analyzed the use of hESC lines in experimental research published from 2008 to 2016. Inspection of hESC research papers revealed that application and/or derivation of a total of 1,419 different hESC lines was reported from 2008 to 2016. Table 3 shows those 21 hESC lines that were used most frequently in these studies. There was no fundamental change in the usage of hESC lines compared with our earlier studies (Guhr et al., 2006, Löser et al., 2010, Schuldt et al., 2013), as expected on the basis of our power law model. Again, three of the five oldest hESC lines (WiCell H1 [WAe001-A], H7 [WAe007-A], and H9 [WAe009-A]), already published in 1998, were most commonly used. These three hESC lines were used in more than 74% of countries that contributed to hESC research from 2008 to 2016 (32 of 43 countries). However, research from countries in which none of these lines were applied accounted for only 1.5% of total papers. The cell line H9 was used in more than 2,200 studies published from 2008 to 2016 (46.4% of all published original research papers involving experimental use of hESCs), followed by hESC lines H1 (23.5%), H7 (7.4%), HES-3 (ESIBIe003-A, 6.0%), HUES9 (HVRDe009-A, 3.8%), and BG01 (VIACe001-A, 3.6%). With the notable exception of HUES9, which was only published in 2004, these hESC lines as well as five other lines among these top 21 ( H14 [WAe014-A], HES-2 [ESIBIe002-A], HSF-1 [UCSFe003-A] and HSF-6 [UCSFe002-A], and H13 [WAe013-A]) were already available to NIH-funded US researchers before the change in the US stem cell policy under the Obama administration, supporting our earlier notion that this political shift had little impact on established stem cell usage patterns.

Table 3

Most Frequently Used hESC Lines

hESC Line	hPSCreg Nomenclature	2008–2010		2011–2013		2014–2016		Total (2008–2016)
		Number of Papers	% of Papers	Number of Papers	% of Papers	Number of Papers	% of Papers	Number of Papers	% of Papers
H9	WAe009-A	495	41.5	847	48.4	860	47.8	2202	46.4
H1	WAe001-A	289	26.9	411	23.5	414	23.0	1114	23.5
H7	WAe007-A	93	8.7	136	7.8	122	6.8	351	7.4
HES-3	ESIBIe003-A	80	7.4	104	5.9	103	5.7	287	6.0
HUES9	HVRDe009-A	60	5.6	64	3.7	54	3.0	178	3.8
BG01	VIACe001-A	81	7.5	60	3.4	31	1.7	172	3.6
HES-2	ESIBIe002-A	60	5.6	67	3.8	45	2.5	172	3.6
KhES-1	KUIMSe001-A	37	3.4	52	3.0	45	2.5	134	2.8
HUES7	HVRDe007-A	31	2.9	35	2.0	34	1.9	100	2.1
KhES-3	KUIMSe003-A	24	2.2	47	2.7	25	1.4	96	2.0
HSF-6	UCSFe002-A	44	4.1	31	1.8	17	0.9	92	1.9
H14	WAe014-A	27	2.5	35	2.0	23	1.3	85	1.8
HUES6	HVRDe006-A	16	1.5	33	1.9	26	1.4	75	1.6
HUES1	HVRDe001-A	28	2.6	22	1.3	17	0.9	67	1.4
HUES3	HVRDe003-A	23	2.1	26	1.5	17	0.9	66	1.4
HUES8	HVRDe008-A	17	1.6	20	1.1	28	1.6	65	1.4
HS181	KIe001-A	29	2.7	20	1.1	12	0.7	61	1.3
HSF-1	UCSFe003-A	25	2.3	23	1.3	8	0.4	56	1.2
MEL-1	SCSe001-A	12	1.1	24	1.4	18	1.0	54	1.1
CA1	MSHRIe001-A	16	1.5	24	1.4	10	0.6	50	1.1
H13	WAe013-A	17	1.6	23	1.3	8	0.4	48	1.0

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Shown are the numbers of papers that report on experimental use of the particular hESC line. The percentage values present the share of publications in the total number of hESC research papers in the given period. Only results for cell lines that were used in at least 1% of papers published from 2008 to 2016 are shown. Please note that in many studies more than one hESC line was used. Sublines and (genetically modified) derivatives were allocated to the respective parental hESC line. hESC lines that were derived before August 22^nd 2001 are marked with asterisks. hPSCreg, Human Pluripotent Stem Cell Registry of the European Union.

To examine whether the predominant use of only a few well-characterized hESC lines is mirrored by a possible long-lasting decrease in the derivation of novel hESC lines, we next determined the number of hESC lines derived from IVF embryos and published during the 9-year period from 2008 to 2016. hESC lines that were produced from nuclear transfer embryos or entities derived by parthenogenetic activation of human oocytes were not included in our analyses. By November 2009, 1,071 such original hESC lines were publicly known, and nearly 694 (64.8%) of them were published in peer-reviewed English-language journals listed in the PubMed database (Löser et al., 2010). By 2016, at least 2,168 original hESC lines were publicly known, 1,544 (71.2%) of which were published in peer-reviewed journals. To unveil possible trends in the time course of hESC derivation, we inspected hESC research papers published from 2008 to 2016 for reports on derivation of novel hESC lines or on the use of (novel) hESC lines that were not used in previously published research, respectively. The results of the analysis are shown in Figure 3. Although 566 novel hESC lines were reported in the scientific literature published from 2008 to 2010, this number decreased to 301 and 220 for the following 3-year periods 2011 to 2013 and 2014 to 2016, respectively (Figure 3A). Notably, an increasing percentage of the novel lines were either derived from pre-implantation genetic diagnosis embryos to model genetically inherited diseases (disease-specific hESC lines) or were produced for future clinical applications (clinical-grade hESC lines; Figure 3B). Thus, the availability of well-characterized hESC lines that are accepted and broadly used by the scientific community seems to cause a decreasing interest in deriving novel research hESC lines, and obviously new hESC lines are produced increasingly for specific scientific and future clinical applications.

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Figure 3

Derivation of New hESC Lines (2008–2016)

(A) Number of hESC lines that were reported for the first time in original research papers during the time periods indicated.

(B) Percentage of disease-specific (light blue) and clinical-grade (dark blue) hESC lines in total new cell lines reported in original research papers during the time periods indicated.

Discussion

Nearly 20 years after the first publication of the successful derivation of hPSCs, we report a sustained interest in hESCs and strongly increasing research activities with hiPSCs. Our findings are based on a manually curated database of experimental hPSC papers that mirrors the genuine research in this field. The manual curation avoids the risk of the presence of non-relevant papers usually found in data pools based on mere and partially automatized web searches, which may result in over-estimation of real research activities (Kobold et al., 2015).

Our analysis of the hPSC landscape shows that research involving hESCs and/or hiPSCs is still increasing, albeit with a much faster increase for the hiPSC field. While this trend can be easily anticipated for hiPSCs, a continuing interest in hESC research cannot necessarily be expected. hESCs are more difficult to derive than hiPSCs and are not as easily accessible as hiPSCs for researchers. Consequently, only comparatively few laboratories have their own hESC lines. Moreover, research in hESCs is controversial for ethical reasons and legally tightly regulated in many countries. For example, in Germany, researchers who plan to use hESCs have to show that hESCs cannot be replaced by hiPSCs in their research project before hESC usage will be approved. Despite this, our data consolidate the previous finding, that research in hESCs and hiPSCs still existed independently and only partially overlapped in recent years. Moreover, use of hESC as a mere “gold standard” control for hiPSC research (which was speculated to be the main area of application for hESCs after emergence of hiPSCs) remains a rather small area.

We also detected some changes in regional distribution of hPSC research. Although US-based scientists considerably contributed to hPSC work published in recent years, the share of US-based research in worldwide research is decreasing. In contrast, especially Chinese groups increasingly published in the hESC field, while scientific groups from Germany increasingly contributed to worldwide hiPSC research. The growing performance of groups from these two countries in hESC and hiPSC research, respectively, may be an immediate consequence of extensive funding programs and strong political support of these particular types of research.

A general look at the impact of hESC research reveals a gradual growth of the overall average impact factor from 6.030 in 2005 (Guhr et al., 2006) to 7.422 in 2009 (Löser et al., 2010) and 7.749 in the present study. In recent years, research with the highest impact was published by researchers from Canada, the Netherlands, the United States, and Israel, confirming their position from previous years, while research from Korea, Singapore, and China underperformed. In addition to real quality differences of research, a possible publication bias in English-language journals, especially for groups from Asian countries, may explain this observation.

Also confirming earlier findings is the high divergence of the average citation numbers of papers from different countries, which is a more reliable measure of the actual relevance of a given publication. Again, we found a predominance of papers from countries such as Canada, the US, the Netherlands, the UK, and Israel with respect to their average citation numbers, while the above-average position of hESC papers from Germany is a rather unexpected result since Germany has relatively restrictive hESC legislation. Interestingly, we noted an average underperformance of research from Japanese and German groups with respect to impact in the hiPSC field. This is difficult to explain, especially for Japan. For this reason, we repeated the citation analysis using the Scopus database (Elsevier), which confirmed our results in general (data not shown). One possible explanation for this phenomenon could be the comparatively broad and generous funding policy with respect to hiPSC (but not to hESC) research in both countries, which might also promote less competitive research performed by less experienced scientists. This could possibly reduce the average quality of research, resulting in a diminished average impact of publications. However, such a broad funding policy will most likely open hiPSC research to scientists new in the field who may contribute substantially to the scientific performance of these countries in the future.

When looking at the hESC lines actually used in published research, our data confirm former results with respect to hESC usage patterns of only a handful of established lines as predicted by our power law model (Schuldt et al., 2013). Overall, although research with hESC is not decreasing, the derivation of new lines is regressive. This is likely due to the existence and widespread use of well-characterized established lines such as H1, H7, and H9, and to the emergence of hiPSCs. The use of only few lines in a large percentage of hESC studies may be of advantage with respect to the comparability and reproducibility of results. On the other hand, the dominance of only a few hESC lines in research is accompanied by a lack of universal applicability of results as well as by genetic under-representation and dependence on only a few suppliers.

Additionally, our findings are in good agreement with data obtained from the hPSC registry of the European Union (hPSCreg, https://hpscreg.eu) (Seltmann et al., 2016). According to the data in hPSCreg, the generation of new hESC lines increased and peaked in the 2005 to 2007 period, after which the numbers of newly derived lines started to decrease. We found that a large portion of hESC lines published in the past decade were generated for specific purposes such as to provide disease models or to generate clinical-grade lines. We are aware that the actual number of hESC lines cannot be determined since only limited information is publicly available. In this study, we therefore focused on hESC lines published in the peer-reviewed scientific literature since we consider this a reliable measure for quality and accessibility of hESC lines. It should also be noted that there is frequently a gap between derivation and publication dates of hESC lines. Consequently, our data reflect the time points of availability of new hESC lines to the scientific community rather than the dates of their derivation.

A quantitative assessment of trends in hiPSC derivation is almost impossible. We noticed that the increasing convenience of hiPSC derivation has caused many laboratories to establish their own hiPSCs. However, in many cases, only limited information on the exact number and the degree of characterization of derived hiPSC lines is provided in published research. In addition, the naming of lines is frequently ambiguous and, in a range of cases, well-characterized lines and picked clones cannot be discriminated from the data presented in the respective papers. A recently proposed standard nomenclature for hPSC lines would be required to trace the cells and their data (Kurtz et al., 2018). However, based on our analysis of more than 3,500 hiPSC research papers, we estimate that the number of hiPSC lines published in the literature up to now exceeds 10,000 by far, many of them derived from patients suffering from genetic diseases. In addition, there are commercial and academic hiPSC banks that host thousands of hiPSC lines, although the degree of characterization of specific cell lines is not clear in many cases (De Sousa et al., 2017). hPSCreg currently registers approximately five times more hiPSC than hESC lines, reflecting the ratio and an increasing number of hiPSC lines versus a rather stable number of registered hESC lines (S.S. et al., unpublished data).

One of the major promises of hPSC is their clinical application in regenerative therapies. We provide a comprehensive overview of clinical trials that have been performed so far based on hESC- and hiPSC-derived cell products. Our data are based on an intensive long-term observation of the field and collection of publicly available information over the past 7 years. However, it should be noted that some of the clinical trials listed in Table 4 are performed under the clinical research study pathway, which may not directly lead to a new medical product marketing registration. These and other studies, which are performed in the frame of specific “regenerative medicine” regulatory frameworks, have the potential to speed up the clinical translation of hPSC research in an academic setting but may not directly result in applicable cell products. This is different for the often commercially driven clinical trials that are performed under the formal "clinical trial" pathway supervised by the national drug regulatory agencies. It should also be noted that simply searching clinical trial registries for the term “hiPSC” usually results in many hits. However, an analysis of the respective trials reveals that most of them only refer to the production of clinical-grade hiPSC lines from specific patients but not to a treatment of patients with hiPSC-derived therapeutic cells. These studies were therefore not included in our results, but it shows that a dedicated clinical trial registry for hPSC-based studies is highly desirable for ethical reasons to provide transparency and avoid replication (Fortunato et al., 2018). Our data show that most studies performed so far use hESCs, most likely because these cells have been available for a longer period, and their derivation is not complicated by additional reprogramming manipulations. The hESC-based clinical studies are performed with very few, mostly older lines. However, we also found publicly available information on at least 11 additional planned hPSC-based studies, four of them using hESCs. Since newer, clinical-grade hESC lines are available now, it will be interesting to see whether hESC usage patterns will be sustained in clinical studies as well, or whether these new lines will replace older lines quickly.

One of the major advantages of hiPSCs is the potential for autologous application. However, the only study using autologous hiPSC lines was put on hold because of mutations of unknown potential risk in the hiPSC source material (Garber, 2015). This study was continued with allogeneic, HLA-matched hiPSCs. Whether autologous, personalized hiPSCs, or HLA-haplobanks of hiPSC lines, will become an affordable option for personalized medicine remains disputable (Blair and Barker, 2016). Since HLA matching will not be perfect in the majority of cases, lifelong immunosuppression may be unavoidable. Unless affordable technologies for autologous clinical hiPSC use will be developed, their allogenic use will most likely diminish the effects of the biggest advantage of hiPSCs over hESCs for cell therapy applications.

In conclusion, we have shown a continuing parallel research at high level and impact with hESCs and hiPSCs, while the increase in published research with hiPSCs accelerates faster than that of hESC research, surpassing those by 2015. While hESC usage patterns remained unchanged over the past decade, the derivation of new hESC lines for disease modeling and clinical application may foreshadow a similar trend for hiPSC research, although data to assess these tendencies are difficult to obtain and would need centralized resources, common standards for characterization, and traceable nomenclature. However, we expect that the hiPSC usage patterns will be less pronounced than that observed for hESC lines in research since hiPSCs are much easier to obtain. As the field is consolidating, we predict a trend toward a more diverse source of hESC and hiPSC lines required for tailored applications; e.g., to reflect human and disease diversity, or to improve safety by using dedicated clinical-grade lines in clinical trials.

Experimental Procedures

Author Contributions

A.G., S.K., S.S., and P.L. performed the collection and assembly of data. A.E.M.S.W., A.K., and P.L. interpreted the data. A.G., A.E.M.S.W., A.K., S.K., S.S., and P.L., performed the data analysis. A.E.M.S.W., A.K., and P.L. conceived and designed the study and wrote the manuscript.

Acknowledgments

Notes

Published: July 19, 2018

Footnotes

References