Enzymatic Passaging Research Articles

214 Different pluripotency maintenance supplements affect the reprogramming process and pluripotency state of bovine-induced pluripotent stem cells

After the emergence of induced cell reprogramming, achieved through the addition of Yamanaka transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) to somatic cells, the number of studies regarding induction and maintenance of pluripotency has increased greatly. The success of bovine iPSCs (biPSCs) was first described by Summer et al. (2011 J. Anim. Sci. 89, 2708-2716; https://doi.org/10.2527/jas.2010-3666); however, investigations on the pluripotent state of biPSCs are still needed because different protocols and characterisation profiles have since been used. The aim of this study was to produce biPSC lines supplemented with different pluripotency maintenance agents to improve self-renewal and pluripotency maintenance. For that, bovine fetal (50 days) fibroblasts (3×104) were transduced with lentivirus harbouring mouse OSKM transcription factors. The cells were further cultured in reprogramming medium (Dulbecco's modified Eagle's medium/F12 KO and 20% KSR (knockout serum replacement)) supplemented with basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), bFGF+2i or LIF+2i (where 2i inhibitors are PD0325901 and CHIR99021). The capacity for cell reprogramming was analysed by colony formation and maintenance after manually and enzymatic passaging and alkaline phosphatase (AP) activity detection; additionally, pluripotency state was assessed by reverse transcription (RT)-PCR (pluripotency biomarkers: OCT4, NANOG, and SOX2; naïve state: STELLA, LIFr, and ESRRb; primed state: OTX2 and FGF5; and mouse (m)OSKM and invitro differentiation assay (embryoid body formation). Statistical analysis was performed using the JMP software (SAS Institute Inc.). All treatments were successful at generating colonies after 28 days of mOSKM transduction, with 32 colonies in bFGF (0.53% efficiency), 21 colonies in bFGF+2i (0.35% efficiency), 5 colonies in LIF (0.08% efficiency), and 3 colonies in LIF+2i (0.05% efficiency) treatments/groups. As an initial pluripotency test, all colonies were positive for AP activity at passage 3. The colonies were cultured for at least 25 passages (±200 days) except for those from the LIF+2i treatment, which were not able to remain viable after 15 passages. Gene expression analysis of the pluripotency (naïve and primed) biomarkers in biPSCs by RT-PCR revealed that colonies from the bFGF treatment were upregulated in NANOG, OCT4, (pluripotency biomarkers), and STELLA (naïve biomarker) (P<0.05) compared with bFGF+2i and LIF groups. There were no differences in expression of SOX2 (pluripotency biomarker gene) and naïve/primed biomarkers (OXT2, LIFr, and ESRRb) (P>0.05). Additionally, the relative abundance of mOSKM was not different between groups (P>0.05). For further pluripotency analysis, biPS colonies were tested for the invitro differentiation assay, and all colonies tested were able to form embryoid bodies. In conclusion, bovine fetal fibroblasts were successfully reprogrammed when using OSKM in all medium tested; however, LIF+2i treatment did not grow beyond 25 passages. Further tests should be performed to determine the pluripotency status of these biPSCs. We acknowledge FAPESP for funding (grant nos. 2012/50533-2, 2015/26816-5, and 2016/16841-2).

Tyroxine Hydroxylase-Positive Neuronal Cell Population is Increased by Temporal Dioxin Exposure at Early Stage of Differentiation from Human Embryonic Stem Cells.

Background: The neurological effects of short-term dioxin exposure during the fetal period is an important health risk in humans. Here, we investigated the effects of dioxin on neural differentiation using human embryonic stem cells (hESCs) to evaluate human susceptibility to dioxin. Methods: Using an enzymatic bulk passage, neural differentiation from human ESCs was carried out. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was added to various stages of culture. The expression levels of the neuronal markers microtubule-associated protein 2 (MAP2) and thyroxine hydroxylase (TH) were measured by RT-qPCR and image analysis of immunostaining. Results: Although early-stage neuronal cells are quite resistant to TCDD, the numbers of neural rosettes and increases in mRNA expression levels and the number of cells positive for MAP2 and TH were significant by temporal exposure at embryoid body stage (Day9-exposure group). In contrast, the TCDD exposures against ESCs (Day0-exposure group) and differentiated neural cells (Day35-exposure group) were not affected at all. The increment was similarly observed by continuous exposure of TCDD from Day9 through Day60. Conclusions: These results indicated that dioxin exposure during the early stage of differentiation from hESCs increases the contents of neuronal cells, especially TH-positive neuronal cells. Regulations of aryl hydrocarbon receptor (AHR) signaling in an early stage of embryogenesis should be investigated extensively to understand the mechanism underlying the increase in neuronal cell populations and to apply the knowledge to regenerative medicine.

Comparison of dissociation methods on pluripotency of stem cells

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance.

To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications.

Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Passaging Techniques and ROCK Inhibitor Exert Reversible Effects on Morphology and Pluripotency Marker Gene Expression of Human Embryonic Stem Cell Lines

Human embryonic stem cells (hESCs) are known for their potential usage in regenerative medicine, but also for handling sensitivity. Much effort has been put into optimizing the culture methods of hESCs. It has been shown that the use of Rho-associated coiled-coil kinase inhibitor (ROCKi) decreases the cellular stress response and the apoptotic cell death in hESC cultures that have been passaged enzymatically. These observations sparked a wide use of ROCKi in hESC cultures. We and others, however, noted that cells passaged enzymatically with the use of ROCKi had a different morphology compared to cells passaged mechanically. Here we show that hESCs that were enzymatically passaged displayed alterations in the nuclear size compared to cultures that were mechanically passaged. Notably, a dramatically decreased expression of the genes encoding common pluripotency markers, such as OCT4/POU5F1 and NANOG were revealed in enzymatically passaged hESCs compared to mechanically passaged, while such differences were not significant when assessing protein levels. The differences in gene expression did not correlate strongly with commonly analyzed histone modifications (H3K4me3, H3K9me3, H3K27me3, and H4K16ac) on the promoters of these genes. Surprisingly, the effects of enzymatic passaging were at least in part reversible as the gene expression profile of enzymatically passaged hESCs that were transferred back to mechanical passaging, showed no significant difference compared to those hESCs that were continuously passaged mechanically. Our results suggest that enzymatic passaging influences parameters associated with hESC characteristics, and emphasizes the importance of using cells handled in the same manner when comparing results both within and between projects.

Human embryonic fibroblasts support single cell enzymatic expansion of human embryonic stem cells in xeno-free cultures

The future application of human embryonic stem cells (hESC) for therapeutic approaches requires the development of xeno-free culture conditions to prevent the potential transmission of animal pathogens or xenobiotic substances to hESC. An important component of the majority of hESC culture systems developed is the requirement for fibroblasts to serve as feeders. For this purpose, several studies have used human foreskin fibroblasts established under xeno-free conditions. In this study we report xeno-free establishment and maintenance of human embryonic fibroblasts (XHEF) and demonstrate their ability to support long-term self-renewal of hESC under xeno-free culture conditions, using a commercially available complete medium. Importantly, our culture conditions allow enzymatic passaging of hESC. In contrast, hESC cultured on human foreskin fibroblasts (XHFF) under the same conditions were poorly maintained and rapidly subject to differentiation. Our study clearly shows that the source of human fibroblasts is essential for long-term xeno-free hESC maintenance.

Culture Conditions and Enzymatic Passaging of Bovine ESC-Like Cells

Culture Conditions and Enzymatic Passaging of Bovine ESC-Like Cells

Culture Conditions and Enzymatic Passaging of Bovine ESC-Like Cells

The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p < 0.05), and bESC-like cells differentiated quickly when treated with Liberase Blendzyme 3. The bESC-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as SSEA-1, AP, OCT-4, and Nanog. When removed from feeders, these bESC-like cells formed embryoid bodies (EBs) in a suspension culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.

Gene Targeting in a HUES Line of Human Embryonic Stem Cells Via Electroporation

Genetic modification is critical for achieving the full potential of human embryonic stem (ES) cells as a tool for therapeutic development and for basic research. Targeted modifications in human ES cells have met with limited success because of the unique culture conditions for many human ES cell lines. The HUES lines of human ES cells were developed for ease of manipulation and are gaining increased utility in stem cell research. We tested conditions for gene targeting via electroporation in the HUES-9 human ES cell line and demonstrate here successful gene targeting at the gene encoding Fezf2 (also known as Fezl), a transcription factor involved in corticospinal neuron development. With a targeting strategy involving positive and negative selection that is applicable to all genes, we observed a gene targeting frequency of approximately 1.5% for Fezf2, a gene not expressed in human ES cells. We found that conditions developed for gene targeting in mouse ES cells can be readily adapted to HUES cells with few key modifications. HUES-9 cells exhibit an intrinsically high efficiency of clonal expansion and sustain electroporation-based gene targeting procedures without any significant loss of pluripotency marker expression or karyotypic stability. Thus, human ES cell lines adapted for enzymatic passage and efficient clonal expansion can be highly amenable to genetic modifications, which will facilitate their application in basic science and clinical development.

Expansion of Human Embryonic Stem Cells In Vitro

This unit describes a protocol for the large-scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes.

Expansion of Human Embryonic Stem Cells In Vitro

AbstractThis unit describes a protocol for the large‐scale expansion of karyotypically normal human embryonic stem cells (hESCs). hESCs can be maintained indefinitely as dense colonies that are mechanically cut into pieces, which are subsequently transferred to fresh organ culture dishes seeded with primary mouse embryonic fibroblasts (MEFs). hESCs can also be enzymatically passaged (bulk culture); however, over time, this style of culturing may lead to the acquisition of chromosomal abnormalities. Nevertheless, enzymatic passaging can be used for short periods (up to 25 passages) without the appearance of cells with abnormal karyotypes. Curr. Protoc. Stem Cell Biol. 1:1C.1.1‐1C.1.7. © 2007 by John Wiley &amp; Sons, Inc.

Automated Mechanical Passaging: A Novel and Efficient Method for Human Embryonic Stem Cell Expansion

There is a need for more standardized methods of maintenance and propagation of human embryonic stem cell (hESC) cultures. Enzymatic passaging currently represents the most widely used method for expansion of hESCs. Although rapid and straightforward, this technique results in variable-sized cell clusters and significant cellular trauma, which may apply selective pressure in long-term culture. Mechanical passaging has the potential advantages of defined colony fragment sizes, reduced cellular trauma, and the possibility of selecting undifferentiated colonies for transfer. However, manual dissection of individual colonies is a prohibitively time-consuming process unsuitable for maintaining large numbers of hESCs without the use of additional chemical means. In this study we report an efficient automated method for mechanically passaging hESCs. We have used this method exclusively to maintain hESCs in long-term undifferentiated culture without the use of enzymatic digestion for longer than 100 days. This automated technique can thus be used routinely to culture hESCs in the laboratory.

Microcarrier cell culture of neonatal human corneal endothelium

Fresh isolates of neonatal human corneal endothelial cells were maintained in tissue culture using a technique employing collagen-coated, dextran-based microcarrier beads. This method provides large yields of endothelial cells suitable for biochemical and/or transplant studies without exposing the cells to enzymatic passage. Our results suggest that microcarrier culture of human corneal endothelial cells can provide a useful in vitro system for the growth and maintenance of actively mitotic cells.