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. 2009 Feb;42(1):63-74.
doi: 10.1111/j.1365-2184.2008.00571.x.

Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation

S M Prasad  1 , M Czepiel, C Cetinkaya, K Smigielska, S C Weli, H Lysdahl, A Gabrielsen, K Petersen, N Ehlers, T Fink, S L Minger, V Zachar
Affiliations

Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation

S M Prasad et al. Cell Prolif. 2009 Feb.

Abstract

Objective: The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, hESCs have a propensity to differentiate spontaneously. In this study, we assessed the nature of hESC responses to hypoxic conditions.

Materials and methods: Human embryonic stem cells were grown in normoxic and hypoxic conditions, and the cells expressing Oct4 and stage-specific embryonic antigen-1 were identified by indirect immunofluorescence. The transcriptional expression of Nanog, Notch1, and Oct4 was determined by a real-time reverse transcription-polymerase chain reaction, and the inhibition of Notch-mediated signalling was achieved with a gamma-secretase inhibitor.

Results: In contrast to culture at 21% oxygen, where the colonies displayed a marked degree of differentiation, we found that during exposure to 5% oxygen, the hESC colonies displayed a homogenous and flat morphology that was consistent with the presence of Oct4-positive phenotype, indicating no spontaneous differentiation. When cultured at 5% oxygen for either 4 weeks or up to 18 months, high levels of Nanog and Notch1 transcriptional expression were detected, albeit the expression was significantly lower during longer exposure. The suppression of differentiation was rapidly reversed on transfer of the hypoxic cultures to normoxic conditions. Looking into the molecular mechanisms of the maintenance of self-renewal at low oxygen tensions, we found that inhibition of Notch signalling fully abrogated the hypoxic induction of undifferentiated phenotype.

Conclusion: Our data, thus, indicate that hypoxic exposure has the capacity to sustain long-term self-renewal of hESCs and that this effect is mediated through activation of Notch.

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Figures

Figure 1
Figure 1
The effect of short‐ and long‐term hypoxic exposure and re‐oxygenation on the morphology of hESC colonies. (a) The CLS1 line was sub‐cultured by mechanical dissection and grown for a period of 4 weeks in five different concentrations of atmospheric oxygen, covering the range from 1% to 20%. After the initial treatment, colonies from all conditions were grown at oxygen concentration of ambient air for additional 4 weeks. (b) The CLS1‐LT line has been continuously maintained in hypoxic conditions corresponding to 5% oxygen for over 18 months. The development of colony morphology between regular passaging intervals is shown over the course of 4 weeks. The response to re‐oxygenation corresponding to the oxygen concentration of ambient air was followed during the course of additional 4 weeks. For each gaseous condition, the development of a representative colony is shown. The images were taken with a maximum zoom, given the size of a particular colony, and stereo microscope illumination settings were adjusted to a dark field observation. Image scalings at the bottom line images apply to all images within the specific week. hESC, human embryonic stem cell.
Figure 2
Figure 2
The effect of short‐term hypoxic treatment and re‐oxygenation on the frequency of spontaneous differentiation of hESCs. (a) The CLS1 line was sub‐cultured by microdissection and grown for a period of 4 weeks at four different levels of hypoxia, covering the range from 1% to 15% of oxygen in the atmosphere. The undifferentiated zones were revealed through the expression of Oct4 (green), the areas of differentiation by detection of SSEA1 (red), and nuclei were counterstained with Hoechst 33342 (blue). The progress of spontaneous differentiation was determined by comparing the extent of Oct4‐positive areas to the total surface of respective colonies. (b) After having been grown for a period of 4 weeks in hypoxic conditions, the CLS1 line was passaged and transferred to oxygen concentration of ambient air. The analysis of spontaneous differentiation was done as with hypoxic cultures (a). The images are mosaics of 20 single fields at 2.5‐fold magnification, and the scale bars correspond to 4 mm. Each point on the graphs represents an average of two independent experiments, where error bars denote standard error of mean. Asterisk indicates a significant difference (P < 0.05) between hypoxic culture at 5% and control normoxic culture 20% oxygen. SSEA, stage‐specific embryonic antigen; hESC, human embryonic stem cell.
Figure 3
Figure 3
The effect of long‐term hypoxic treatment and re‐oxygenation on the frequency of spontaneous differentiation within the colonies of hESCs. Prior to this experiment, the CLS1‐LT line was maintained in hypoxia of 5% oxygen for over 18 months. (a) After sub‐culturing by mechanical microdissection, and further growth in identical hypoxic conditions or replacing into normoxic conditions corresponding to ambient air, the undifferentiated zones were revealed through the expression of Oct4 (green), the areas of differentiation by detection of SSEA1 (red), and nuclei were counterstained with Hoechst 33342 (blue). The images are mosaics of 144 single fields at 10‐fold magnification, and the scale bars correspond to 1 mm. (b) The progress of spontaneous differentiation was determined from the ratio of Oct4‐positive areas to the total surface of respective colonies. The plots are based on averages from two independent experiments and error bars denote standard error of mean. Asterisk indicates a significant difference (P < 0.05). SSEA, stage‐specific embryonic antigen; hESC, human embryonic stem cell.
Figure 4
Figure 4
Structure of hESC colonies maintained at ambient air oxygen concentration or exposed to moderate hypoxia during short‐ or long‐term culture. CLS1 line was routinely grown in the presence of 20% oxygen, and for short‐term hypoxic exposure it was cultured in 5% of oxygen atmosphere for 3 weeks. CLS1‐LT cell line, that has previously been maintained in long‐term (>18 months) hypoxic conditions corresponding to 5% oxygen, has also been sub‐cultured in 5% oxygen for 3 weeks. The nuclei are rendered in blue (Hoechst 33342), undifferentiated areas in green (Oct4+), and differentiated areas in red (SSEA1+). Arrowheads indicate accumulations of cells negative for both Oct4 and SSEA1. The overview images are mosaics, entailing from 30 to up to 120 single fields, at 10‐fold magnification. SSEA, stage‐specific embryonic antigen; hESC, human embryonic stem cell.
Figure 5
Figure 5
Kinetics of expression of Oct4‐, Nanog‐, and Notch1‐specific messages in hESCs during short‐ and long‐term hypoxic exposure by real‐time RT‐PCR. The CLS1 and CLS2 hESCs lines were grown in the normoxic conditions of ambient air or in short‐term hypoxia of 5% oxygen concentration. CLS1‐LT and CLS2‐LT cell lines, that have previously been maintained in long‐term (>18 months) hypoxic conditions corresponding to 5% oxygen, have also been sub‐cultured in 5% oxygen for 4 weeks. For the analysis in normoxic conditions, the differentiated and undifferentiated parts of the colonies were separated from feeder layer. In hypoxic conditions, whole, feeder‐free, colonies were analysed. The expression levels were normalized to the undifferentiated cells from control cultures at the first week. The error bars denote standard error of mean and they are shown only in positive direction. Asterisks indicate a significant difference (P < 0.05). undif, undifferentiated; dif, differentiated; hESC, human embryonic stem cell.
Figure 6
Figure 6
Proliferation of hESCs grown for short‐ or long‐term in hypoxic conditions. The CLS1 cells were routinely grown at 20% oxygen, and exposed to 5% oxygen during a short‐term 4‐week culture. The CLS1‐LT cell line was grown for 4 weeks at 5% oxygen following a previous long‐term (>18 months) propagation in hypoxia. (a) The rate of the colony growth was determined from incorporation of [3H]‐thymidine (left panel) and the relative proliferative activity as a ratio of proliferating areas to the total colony surface was determined from incorporation bromodeoxyuridine (right panel). The plots are based on averages from two independent experiments, and the error bars denote standard error of mean. The asterisk indicates a significant difference (P < 0.05). (b) Microscopic analysis of the proliferation pattern was done both at an overview magnification and a higher resolution at 3 weeks post‐sub‐culturing. The overview images are mosaics of 144 single fields at 10‐fold magnification. ST, short‐term; LT, long‐term; hESC, human embryonic stem cell.
Figure 7
Figure 7
Reversal of undifferentiated hESC phenotype induced by short‐ or long‐term hypoxia through inhibition of Notch1 signalling. The CLS1 line was treated with γSI during a 3‐week normoxic or short‐term hypoxic culture involving 20% or 5% oxygen concentrations, respectively. The cell line CLS1‐LT that has previously been propagated at 5% oxygen in long term (>18 months) was included as well. (a) The extent of undifferentiated zones was revealed through the expression of Oct4 (green), the areas of differentiation by detection of SSEA1 (red), and nuclei were counterstained with Hoechst 33342 (blue). The images are mosaics, comprised of 30 to up to 72 single fields, at 10‐fold magnification. Scale bars indicate 1 mm. (b) The effect of inhibition of Notch1 activation was evaluated from the suppression of the proportion of undifferentiated area versus the total colony surface. The plots are based on averages from two independent experiments and error bars denote standard error of mean. Asterisk indicates a significant difference (P < 0.05). γSI, γ‐secretase inhibitor; ST, short‐term; LT, long‐term; hESC, human embryonic stem cell.
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