Amniocytes can serve a dual function as a source of iPS cells and feeder layers

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA.
Human Molecular Genetics (Impact Factor: 6.39). 03/2011; 20(5):962-74. DOI: 10.1093/hmg/ddq542
Source: PubMed


Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5-7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

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Available from: Annick Doan, May 23, 2014
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    • "This results seem to open an applicative consequence: cells gestational age could, in fact, supports different mesodermal and endodermal lineages regenerative properties, as well as, it may offer the transplantation of different rates of stem/progenitor cells. In this context, it is interesting to recall that amnion-derived cells have been proposed as a superior cell type for the production of induced pluripotent stem cells (iPSC) [63–66]. However, only in vivo experiments will be able to demonstrate the full potential regenerative features of this type of stem cells, as suggested by preclinical studies carried out in different transplantation settings [9, 10, 21, 24, 25]. "
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    ABSTRACT: Stem cells isolated from amniotic epithelium (AECs) have shown great potential in cell-based regenerative therapies. Because of their fetal origin, these cells exhibit elevated proliferation rates and plasticity, as well as, immune tolerance and anti-inflammatory properties. These inherent attitudes make AECs well-suited for both allogenic and xenogenic cellular transplants in animal models. Since in human only at term amnion is easily obtainable after childbirth, limited information are so far available concerning the phenotypic and functional difference between AECs isolated from early and late amnia. To this regard, the sheep animal model offers an undoubted advantage in allowing the easy collection of both types of AECs in large quantity. The aim of this study was to determine the effect of gestational age on ovine AECs (oAECs) phenotype, immunomodulatory properties, global DNA methylation status and pluripotent differentiation ability towards mesodermic and ectodermic lineages. The immunomodulatory property of oAECs in inhibiting lymphocyte proliferation was mainly unaffected by gestational age. Conversely, gestation considerably affected the expression of surface markers, as well the expression and localization of pluripotency markers. In detail, with progression of gestation the mRNA expression of NANOG and SOX2 markers was reduced, while the ones of TERT and OCT4A was unaltered; but at the end of gestation NANOG, SOX2 and TERT proteins mainly localized outside the nuclear compartment. Regarding the differentiation ability, LPL (adipogenic-specific gene) mRNA content significantly increased in oAECs isolated from early amnia, while OCN (osteogenic-specific gene) and NEFM (neurogenic-specific gene) mRNA content significantly increased in oAECs isolated from late amnia, suggesting that gestational stage affected cell plasticity. Finally, the degree of global DNA methylation increased with gestational age. All these results indicate that gestational age is a key factor capable of influencing morphological and functional properties of oAECs, and thus probably affecting the outcome of cell transplantation therapies.
    Full-text · Article · May 2014 · Stem Cell Reviews and Reports
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    • "Following the footsteps of two pioneering studies on generating hiPSCs lines [28], [29], hiPSCs are conventionally maintained on MEF feeder cells with 20% KOSR supplemented with a wide range of bFGF, 4–100 ng/ml [30], [31]. A number of studies have also described the successful culture of hiPSCs on human adult feeder cells supplemented with 4–20 ng/ml bFGF [19], [32], [33], [34], [35], [36], [37]. In addition, several feeder-free systems applicable for hESCs culture have also been shown to support hiPSCs culture, including TeSR1 or E8 medium on matrigel, vitronectin, synthetic peptide substrate or laminin [27], [38], [39], [40], [41], StemPro hESC SFM on Geltrix [37], and FTDA medium (0.5 ng/ml TGFβ1, 5 ng/ml Activin A, 50 nM Dorsomorphin, 10 ng/ml bFGF and others) on matrigel [41]. "
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    ABSTRACT: Many different culture systems have been developed for expanding human pluripotent stem cells (hESCs and hiPSCs). In general, 4-10 ng/ml of bFGF is supplemented in culture media in feeder-dependent systems regardless of feeder cell types, whereas in feeder-free systems, up to 100 ng/ml of bFGF is required for maintaining long-term culture on various substrates. The amount of bFGF required in native hESCs growth niche is unclear. Here we report using inactivated adipose-derived human mesenchymal stem cells as feeder cells to examine long-term parallel cultures of two hESCs lines (H1 and H9) and one hiPSCs line (DF19-9-7T) in media supplemented with 0, 0.4 or 4 ng/ml of bFGF for up to 23 passages, as well as parallel cultures of H9 and DF19 in media supplemented with 4, 20 or 100 ng/ml bFGF for up to 13 passages for comparison. Across all cell lines tested, bFGF supplement demonstrated inhibitory effect over growth expansion, single cell colonization and recovery from freezing in a dosage dependent manner. In addition, bFGF exerted differential effects on different cell lines, inducing H1 and DF19 differentiation at 4 ng/ml or higher, while permitting long-term culture of H9 at the same concentrations with no apparent dosage effect. Pluripotency was confirmed for all cell lines cultured in 0, 0.4 or 4 ng/ml bFGF excluding H1-4 ng, as well as H9 cultured in 4, 20 and 100 ng/ml bFGF. However, DF19 demonstrated similar karyotypic abnormality in both 0 and 4 ng/ml bFGF media while H1 and H9 were karyotypically normal in 0 ng/ml bFGF after long-term culture. Our results indicate that exogenous bFGF exerts dosage and cell line dependent effect on human pluripotent stem cells cultured on mesenchymal stem cells, and implies optimal use of bFGF in hESCs/hiPSCs culture should be based on specific cell line and its culture system.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "For clinical applications, quality-controlled xenobiotic-free culture systems are required to minimize health risks from animal-derived pathogens and immunogens [16], [17]. Therefore, the use of primary human-derived living cells, like fibroblasts [18]–[21] or amnion-derived cells [22], is a hopeful approach, although some difficulties with these methods must still be overcome. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived material for maintaining hiPSCs/hESCs [23]. "
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    ABSTRACT: Human ES cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are usually generated and maintained on living feeder cells like mouse embryonic fibroblasts or on a cell-free substrate like Matrigel. For clinical applications, a quality-controlled, xenobiotic-free culture system is required to minimize risks from contaminating animal-derived pathogens and immunogens. We previously reported that the pericellular matrix of decidua-derived mesenchymal cells (PCM-DM) is an ideal human-derived substrate on which to maintain hiPSCs/hESCs. In this study, we examined whether PCM-DM could be used for the generation and long-term stable maintenance of hiPSCs. Decidua-derived mesenchymal cells (DMCs) were reprogrammed by the retroviral transduction of four factors (OCT4, SOX2, KLF4, c-MYC) and cultured on PCM-DM. The established hiPSC clones expressed alkaline phosphatase, hESC-specific genes and cell-surface markers, and differentiated into three germ layers in vitro and in vivo. At over 20 passages, the hiPSCs cultured on PCM-DM held the same cellular properties with genome integrity as those at early passages. Global gene expression analysis showed that the GDF3, FGF4, UTF1, and XIST expression levels varied during culture, and GATA6 was highly expressed under our culture conditions; however, these gene expressions did not affect the cells' pluripotency. PCM-DM can be conveniently prepared from DMCs, which have a high proliferative potential. Our findings indicate that PCM-DM is a versatile and practical human-derived substrate that can be used for the feeder-cell-free generation and long-term stable maintenance of hiPSCs.
    Full-text · Article · Jan 2013 · PLoS ONE
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