This work describes an amniocyte-based stem-cell derivation and culture system that has potential utility for the translational application of human stem-cell-based therapies (Fig. ). This system has several features that potentially address currently problematic aspects associated with existing approaches to stem-cell therapy. The advances tendered by this work include: (i) the derivation of both iPS and feeder cells from a single pool of donor amniocytes, pre-screened for pathogenic human viruses; (ii) the avoidance of exposure to animal products (serum, feeder cells); (iii) the use of live cell immunostaining for efficient selection of reprogrammed iPS cell colonies; and (iv) the previously described enhanced reprogramming efficiency which human amniocytes exhibit (34
Figure 7. Patient-specific stem-cell therapy using human amniocytes in a xenoculture-free, autologous system. In this model, discarded amniocytes are obtained as a byproduct of routine antenatal screening and can be banked. At an appropriate time, they are cultured (more ...)
The central feature of the system is that both cellular components of the system (i.e. iPS cells and their supporting feeder cells) originate from a single cell source. If these cells originate from a future recipient, then the transferred cells, and any feeder cells that might be co-transferred to a recipient, either inadvertently or on purpose, will be autologous to the recipient. In principle, this would further obviate the potential for an immune reaction. The potential availability of human amniocytes, which can be banked and which are considered a discarded tissue by most institutional review boards (IRBs), thus offers the opportunity for the culture of human stem cells and feeders that are both autologous to the intended recipient. While the banking of fetal amniocytes for future use may seem logistically complex, it should be noted that banking of umbilical cord blood cells for similar purposes has already been implemented clinically.
We show that mitotically inactivated amniocytes can serve as feeder layers for the co-culture and self-renewal of both human iPS and ES cells (Fig. ), thereby circumventing the need for MEF feeders and the accompanying concerns about xenoculture and resultant zoonosis. The transfer of human stem cells that have been cultured with nonhuman animal cell feeder layers and then transplanted into a human recipient falls under the FDA definition of xenotransplantation, whereby any ‘human body fluids, cells, tissues, or organs that have had ex vivo
contact with live nonhuman animal cells, tissues, or organs’ are themselves xenotransplant materials (38
). The concern regarding xenotransplantation is the potential for transmission of recognized, or as yet unrecognized, infectious agents from animals to humans (38
). Because it avoids the use of ‘animal cells, tissues or organs,’ our iPS cell derivation and culture system would not involve xenotransplantation. Our hAdiPS cell derivation protocol also avoids the use of fetal bovine serum by employing KOSR, a serum-free culture medium (see Materials and Methods). In addition, the human iPS cell lines derived here originate from human amniotic fluid cells that are already routinely screened for evidence of infectious agents including hepatitis, human immunodeficiency virus and rubella. The use of an individual's own cells and tissues without exposure to animal cells, and after screening for human infectious agents, should help obviate concerns about zoonosis associated with the use of MEF feeders, and about latent viral infection. These features could reduce the burden of testing for certain transmissable diseases. Another obvious limitation of many current iPS cell derivation approaches is the use of viral vectors for efficient derivation. However, research in many laboratories is directed towards developing efficient, viral-free derivation protocols. Although we employed retroviral vectors in the current study, we found it possible to avoid the use of serum, both in iPS cell generation and even in reprogramming vector preparation. Collectively, these improvements should promote clinical translation by improving the overall efficiency of reprogramming from an available clinical source, and by establishing the feasibility of using the same individual's amniocytes as feeders.
It should be noted that other human feeder layers and feeder-free culture media have been described, including defined and serum-free media that support undifferentiated hES cell growth for up to 10 passages (40
). However, hES cells maintained in feederless systems can eventually become karyotypically unstable (42
). Recently, HDFs have been shown to serve as autologous feeders for hiPS cells, but HDF-derived iPS cells and HDF-derived feeders are limited by prolonged derivation times and the fact that not all HDF cell lines efficiently support hES or hiPS cell growth (44
). Nonetheless, the HDF and amniocyte systems may prove complementary, as the former would be useful for the vast majority of who did not have the opportunity to have their amniocytes banked. However, going forward, the large individuals number of amniocenteses performed in the USA and other industrialized nations could provide a practical source for banked amniocytes and for AdiPS cells and feeder cells for cell therapy applications.
Compared to other somatic cell types, amniocytes offer several potential advantages for the generation of iPS cells. Mouse and human amniocytes provide a source of cells that permit the generation of iPS cells with a significantly higher efficiency, by more than 10-fold, relative to MEFs or HDFs. By our most conservative estimate, ~0.5% of mouse or human amniocytes can be reprogrammed to a pluripotent state, as further confirmed by AP and Oct4 expression and by subsequent differentiation experiments (EB formation, teratoma analysis), microarray gene expression and methylation analysis of human iPS cell genomic DNA. In contrast, the efficiencies reported for adult human fibroblasts, MEFs, blood cells, adipose stem cells and keratinocytes are: 0.01, 0.001, 0.001, 0.2 and 0.002%, respectively (4
). Thus, human amniocytes are a highly efficient target cell for iPS cell derivation. Indeed, results from this and other studies (34
) indicate that amniocytes represent the most rapidly reprogrammed cell population by primary infection yet described, with a latency of ~5–6 days, compared with about 10 days to induce iPS cell colonies from keratinocytes (20
) and 2 weeks or more from MEFs (14
). Lastly, our studies in the mouse system suggest that just two factors (Klf4 and c-Myc) are sufficient to initiate pluripotency, and the 2-factor mouse AdiPS cells were able to differentiate into all three germ layers. No other reprogramming protocol that we are aware of permits the omission of Oct4 in iPS cell derivation. Although the use of Klf4 and c-Myc, two oncoproteins, may limit the clinical utility of approaches based on these two factors, similar two factor experiments will be of interest in human amniocytes.
Why are mouse and human amniocytes so readily reprogrammed? One possible explanation relates to the notion that embryonic cells more closely resemble the pluripotent state in terms of their transcriptional and chromatin states than other somatic cell types. Recent work by Hochedlinger and colleagues (27
) using a secondary system to generate iPS cells in the context of hematopoiesis clearly demonstrates that the differentiation state of a cell strongly impacts both the efficiency and kinetics of reprogramming. In particular, progenitor stages for various hematopoietic lineages show higher re-programming efficiencies than more differentiated stages (27
). Thus, while human foreskin fibroblasts and MEFs have been used for iPS cell induction and represent perinatal and embryonic cell types, respectively, amniocytes can be isolated as early as 15 weeks in the human (36
) and at E8.5 in the mouse (our observations). At these early developmental stages, some embryonic cells that are shed into the amniotic fluid may retain substantial multipotency, and may even approximate the pluripotent stem-cell state.
Thus, the observation that amniocytes are so readily reprogrammed may partly reflect their embryonic origin. These cells may already possess a partly pluripotent transcriptional or epigenetic state and be more readily reprogrammed than adult cell types. In this regard, it is notable that while hAdiPS cells demonstrate a highly concordant transcriptional expression profile with hES cells (r2
= 0.98–0.99), non-reprogrammed amniocytes also exhibit significant concordance with hES and hAdiPS cells (r2
= 0.82–0.85) (Supplementary Material, Fig. S3
). In addition, the donor amniocyte OCT4
promoter methylation pattern is heterogenous and incomplete (Fig. M). Although we were unable to show preferential reprogramming of endogenous Oct4
-expressing amniocytes, the incompleteness of Oct4
promoter methylation in amniocytes could facilitate conversion to the demethylated state that characterizes pluripotency. Emerging studies imply that the epigenetic memory of specific donor cell types may restrict the subsequent differentiation potential of derived iPS cells (46
). For example, fibroblast-, hematopoietic- and myogenic-derived iPS cells exhibit distinct transcriptional and epigenetic patterns and differentiation potential (48
). Moreover, iPS cells harbor DNA methylation signatures characteristic of the donor cell, which favors differentiation related to the donor cell and restricts alternative fates (46
). In contrast, by the criteria of in vitro
EB and in vivo
teratoma formation, hAdiPS cells differentiated into tissues representing all three germ layers without obvious preference for a single tissue type or germ layer. The current experiments suggest that amniocytes could be useful for generating iPS cells that can differentiate into a range of cell types.
An extension of this view is that pluripotent or multipotent stem cells already exist within the amniocyte population, and that our iPS cell protocol simply selects for, or reprograms, an existing stem-cell-like population already present in amniotic fluid. Indeed, such an AFS cell population has been described (30
). However, our results show that culture of non-transduced amniocytes does not result in colony formation even with extended culture and more than 30 passages. Similarly, neither AFS cells nor hAFDCs form teratomas in mice (30
), although the requirement for a threshold number of pluripotent cells for teratoma formation (49
) may constrain the sensitivity of this assay. Nonetheless, enrichment for amniocytes that express the c-Kit receptor, which operationally defines AFS cells, does not improve the efficiency of iPS cell induction. Therefore, while our results do not exclude the possibility that a subpopulation of amniocytes contributes to the derivation of AdiPS cell colonies, it seems unlikely that previously described AFS cells in amniotic fluid (30
) preferentially do so. Additional work is required to better understand the heterogeneity of amniocytes and to assess how the different cell types present in amniotic fluid influence the efficiency of iPS cell induction.
Our observations on mouse and human amniocytes are generally in agreement with a recent description of efficient but variable frequency of hiPS cell derivation using human amniocytes that were subjected to long-term culture (>10 passages) and morphologic selection after infection, and then grown on mouse feeder cells (34
). Two key differences between these derivation protocols include the other study's use of morphologic selection prior to analysis, and of mouse feeder layers and serum-containing media for iPS cell derivation. These features of the other protocol may partly explain the over 25-fold variation in iPS cell derivation efficiency between batches of amniocytes that were observed in those studies. In contrast, we employed a serum-free and initially feederless system for hAdiPS cell derivation, and live cell immunoselection for rapid colony purification, prior to long-term propagation on feeders (15
). The use of live cell immufluorescent selection enabled us to clone stem-cell lines with relative ease, and to exclude incompletely reprogrammed clones from further analysis. Lastly, a novel innovation is the finding that amniocytes that are mitotically inactivated with either mitomycin C (for mouse) or γ-irradiation (for human) appear to function with high fidelity for the propagation and maintenance of pluripotent, undifferentiated iPS and ES cells. The amniocyte system thus provides an efficient autologous system for hAdiPS cell generation and culture that could significantly advance the translational application of stem-cell therapy.