Expansion of pluripotent human embryonic stem cells on human feeders

Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications.     

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.     

A feeder-free culture using autogeneic conditioned medium for undifferentiated growth of human embryonic stem cells: Comparative expression profiles of mRNAs,microRNAs and proteins among different feeders and conditioned media

Background  

Human embryonic stem (hES) cell lines were derived from the inner cell mass of human blastocysts, and were cultured on mouse embryonic fibroblast (MEF) feeder to maintain undifferentiated growth, extensive renewal capacity, and pluripotency. The hES-T3 cell line with normal female karyotype was previously used to differentiate into autogeneic fibroblast-like cells (T3HDF) as feeder to support the undifferentiated growth of hES-T3 cells (T3/HDF) for 14 passages.     

Culturing in vitro produced blastocysts in sequential media promotes ES cell derivation

Embryonic stem (ES) cell lines are routinely derived from in vivo produced blastocysts. We investigated the efficiency of ES cells derivation from in vitro produced blastocysts either in monoculture or sequential culture. Zygotes from hybrid F1 B6D2 mice were cultured in vitro to the blastocyst stage in Potassium (K(+)) simplex optimised medium (KSOM) throughout or in KSOM and switched to COOK blastocyst medium on day 3 (KSOM-CBM). Blastocysts were explanted on a feeder layer of mitomycin C-inactivated murine embryonic fibroblasts (MEF) in TX-WES medium for ES cell derivation. Sequential KSOM-CBM resulted in improved blastocyst formation compared to KSOM monoculture. ES cells were obtained from 32.1% of explanted blastocsyts cultured in KSOM-CBM versus 18.4% in KSOM alone. ES cell lines were characterized by morphology, expression of SSEA-1, Oct-4 and alkaline phosphatase activity, and normal karyotype. These results indicate that in vitro culture systems to produce blastocysts can influence the efficiency of ES cell line derivation.     

人孤雌胚胎干细胞在人包皮成纤维细胞饲养层上的生长状态

人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态.     

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts were used, a mechanical method of passaging led to better cell growth than passaging by trypsin digestion. We also found that exogenous supplementation with leukemia inhibitory factor maintained the embryonic stem cell-like cells in an undifferentiated state, whereas addition of stem cell factor resulted in their differentiation. Our findings provide an experimental basis for the establishment of an effective culture system for bovine embryonic stem cells.     

Establishment and characteristics of new human embryonic stem cell subline SC6-FF in an allogenic feeder-free culture system

The novel human embryonic stem cell (hESC) subline SC6-FF was derived from SC6 cells in an allogenic feeder-free culture system. Key components of the feeder-free culture system were extracellular matrix proteins and conditioned medium from the mesenchymal stem cell line SC5-MSC. These conditions are allogenic for SC6-FF cells. SC6-FF subline underwent more than one hundred cell population doublings and retained a normal diploid karyotype; 46, XX. The average population doubling time was 23.7 ± 0.8 h, similar to that of the parent SC6 line. The presence of undifferentiated hESC markers (alkaline phosphatase activity, Oct-4, SSEA-4, and TRA-1-60) was verified by histochemistry and immunofluorescence. Cells were distinguished from parental cells in size and morphology as a result of spontaneous differentiation. These cells exhibited the ability to differentiate into derivates of three germ layers by expressing common markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We could conclude that characteristics of the novel feeder-free SC6-FF subline correspond to the status of human embryonic stem cells.     

Automated,scalable culture of human embryonic stem cells in feeder‐free conditions

Large‐scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor‐intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder‐free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1‐81, and SSEA‐4), stable karyotype, and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell‐use in clinical, scientific, and industrial applications. Biotechnol. Bioeng. 2009;102: 1636–1644. © 2008 Wiley Periodicals, Inc.     

A novel feeder-free system for human embryonic stem cells and characterization of their sublines with autogenic and allogenic cultivation

We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.     

Identification of proteins from feeder conditioned medium that support human embryonic stem cells

The maintenance of undifferentiated human embryonic stem cells (hESC) requires feeder cells, either in co-culture or feeder-free with conditioned medium (CM) from the feeders. In this study, we compared the CM of a supporting primary mouse embryonic feeder (MEF) and an isogenic but non-supporting MEF line (DeltaE-MEF) in order to gain an insight to the differential expression profile of secreted factors. Using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight (MALDI) tandem mass spectrometry, 13 protein identities were found to be downregulated in DeltaE-MEF compared to MEF, of which 4 were found to be soluble factors and 3 proteins were membrane-associated or related to the extracellular matrix. In addition, four other proteins were identified to be differentially expressed in MEF-CM using high pressure liquid chromatography (HPLC) and cytokine arrays. In functional experiments where CM was replaced with six of the factors identified, hESC were able to proliferate for five continuous passages whilst maintaining 68-82% and 74-98% expression of pluripotent markers, Oct-4 and Tra-1-60, respectively. Using proteomic tools, important proteins from CM that supports hESC culture have been identified, which when replaced with recombinant proteins, continue to support undifferentiated hESC growth in a feeder-free culture platform.     

一种新的人胚胎干细胞无饲养层培养方法的建立

目的:以转染碱性成纤维细胞生长因子(bFGF)的人胎肝基质细胞株(FLSC)培养人胚胎干细胞(hESC),寻找更加安全、有效的体外培养扩增方法。方法:通过ELISA方法定量检测转基因的人FLSC条件培养基中bFGF的分泌量;以商业化的mTeSR1无血清无饲养层培养基、常规小鼠胚胎成纤维细胞(MEF)条件培养基,以及转染bFGF的人FLSC条件培养基(bFGF/FLSC-CM)分别培养扩增H9细胞。通过观察hESC形态、免疫荧光染色、流式细胞检测及RT-PCR,检测hESC全能性标志物的表达。结果:ELISA方法检测bFGF/FLSC-CM中bFGF因子的分泌量为(770.09±17.28)pg/mL,而MEF-CM中bFGF因子的分泌量为(55.59±0.61)pg/mL,两者存在显著差异(P0.01);在3种培养体系下,免疫荧光检测hESC全能性标志Oct-4、Tra-1-81抗体的表达均呈阳性,流式检测细胞表面阶段特异性胚胎抗原4(SSEA-4)抗体阳性细胞的比例均在99%左右;RT-PCR检测到hESC特异的转录因子Oct-4、Nanog、Sox-2的表达。结论:以转染bFGF的人FLSC条件培养基可以有效扩增hESC,可为临床应用提供一种安全、高效、低成本的无饲养层培养方法。     

Feeder cell density—A key parameter in human embryonic stem cell culture

Summary A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm², above the recommended value of 20,000 cells/cm², appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm²), the ES cells appear to stack up to form a “bulge.” This was not observed under the recommended feeder cell density of 20,000 cells/cm². By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm². This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.     

Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces

Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on Matrigel(TM). Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing.     

Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.     

Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.