Download New TraNscripTomic sigNaTure of HumaN Dp cells culTureD iN 3D

New Transcriptomic Signature of Human
DP cells cultured in 3D Spheroids by
centrifugation
F. Berthelot(1), T. Pocard(1),
C.A. Higgins(2), C. Tacheau(1),
O. Perin(1), J. Demaude(1)
(1)
L’Oréal Research & Innovation, Aulnay-sous-bois, France
Department of Bioengineering, Imperial College, London, UK
(2)
INTRODUCTION
MATERIALS AND METHODS
Dermal Papilla (DP) cells are specialized mesenchymal cells located in a specific compartment, the Dermal Papilla,
at the bottom of hair follicles. These cells play crucial roles in hair formation, growth and cycling. As Higgins et al. and
Ohyama et al. showed, human DP cells lose their capacity to induce hair morphogenesis when removed from the
follicle and expanded in vitro. By growing DP cells in spheroid cultures, such as hanging drop, to mimic aggregation, a
partial restoration of their inducing activity is observed(1). Thus, DP cells cultured in spheroids could be a more predictive
model for screening of anti-hair loss ingredients. However, hanging drop technique is labor intensive and high throughput screening (HTS) is not possible with this approach. Therefore, we utilised a classical centrifugation process(2,3) to
generate our DP cell spheroids. The aim of this study was to characterize, at the transcriptomic level, this new type
of DP cell spheroids taking into consideration previously published work in order to offer a method that adequately
matches the requirements and needs of HTS test.
RESULTS
4 MOLECULAR PATHWAYS, IMPORTANT FOR HAIR FOLLICLE GROWTH
1 COMPARISON 3D vs 2D IN L’OREAL STUDY
Figure 1: Alkaline phosphatase activity: detected in cells cultured in 2D, but strongly observed in DP
cells spheroid (whole mount), and not detected in Normal Human Dermal Fibroblasts (NHDF), negative
control.
Figure 2: Volcano plot of differentially expressed genes. 4871 genes are differentially regulated between cells cultured in 3D versus cells cultured in 2D
in L’Oréal study (n=3 donors, • p-value < 0.05).
Figure 6: Genes differentially regulated between cells cultured in 3D versus cells cultured in 2D, with genes involved in WNT, BMP and NOTCH signaling pathways highlighted in
pink and genes coding for inhibitors of these pathways highlighted in purple . For each analyzed pathway, the correlation coefficient between 3D vs 2D in L’Oréal and 3D vs 2D in
Higgins studies was calculated (framed value). 3D culture tends to activate WNT signaling within DP cells by inducing expression of the agonist SOX4 and repressing expression
of DKK1, among the strongest antagonist of WNT pathway. 3D culture tends to activate BMP signaling within DP cells mainly by inducing expression of BMP2 and MGP and
repressing expression of CRIM1 and IGFBP3. Culture in 3D structure up-regulated HES1 and HEY1, which suggests activation of NOTCH signaling in DP cells. For these different
molecular pathways, results are correlated between L’Oréal and Higgins studies, in spite of different processes used to generate DP cells spheroids.
2 CORRELATION BETWEEN L’OREAL AND HIGGINS STUDIES
Figure 3: Immunofluorescence analysis of proteins in the in spheroids of DP cells obtained in L’Oréal study
(by centrifugation) and Higgins’ study (1, 4) (by hanging drop).
Like intact dermal papillae, DP cells in spheroid are not mitotically active as indicated by Ki67 or PCNA
expression and expression of periostine (POSTN) is absent in 3D spheroids. Expression of perlecan,
BMP2 and SFRP2 is maintained in dermal spheroid cultures.
Figure 4: Comparison of 3D vs 2D in L’Oréal study and in Higgins study
(p-value < 0.05).
Genes modulated in the same way in both studies
Genes specifically modulated in L’Oréal study
Genes specifically modulated in Higgins study
The Pearson’s correlation coefficient between these 2 studies is of 0.64.
3 BIOLOGICAL PATHWAYS ENRICHED IN THE COMPARISION OF 3D vs 2D
Figure 5: Main biological pathways regulated by switching growth in 2D to growth in 3D are genes coding for cell proliferation, myofibroblast differentiation, angiogenesis signaling
pathways. Similar processes were applied to Higgin’s dataset to allow meta-analyses after merging by gene. For each analyzed pathway, the correlation coefficient between 3D vs 2D in
L’Oréal and 3D vs 2D in Higgins studies was calculated (framed value).
3D culture induces expression of a inhibitor of cell cycle and represses expression of effectors of cell cycle progression. 3D induces expression of inhibitors of myofibroblast differentiation and represses expression of myofibroblast markers. 3D culture of DP cells induced many potent pro-angiogenic genes. These results present strong correlation coefficients
with the Higgins’ study.
References
(1) Higgins CA, Chen JC, Cerise JC, Jahoda CA, Christiano AM. Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair follicle
growth. Proc. Natl Acad. Sci. USA doi:10.1073/pnas.1309970110 (2013).
(2) Ivascu A, Kubbies M. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen. 11:922–932 (2006).
(3) Ohyama M, Kobayashi T, Sasaki T, Shimizu A, Amagai M. Restoration of the intrinsic properties of human dermal papilla in vitro. J Cell Sci 125(Pt 17):4114–4125 (2012).
(4) Higgins CA, Richardson GD, Ferdinando D, Westgate GE, Jahoda CA. Modelling the hair follicle dermal papilla using spheroid cell cultures. Exp. Dermatol. 19(6), 546–548 (2010).
Acknowledgements
We thank S. Desbouis and P. Justine for excellent technical support. We thank K. Alves, L. Buffat, S. De Bernard and S. Tymen, working for Altrabio, a biotechnology company, for integrative
analysis of data obtained in this study.
Figure 7: Genes differentially expressed between cells cultured in 3D versus cells cultured in 2D, with genes involved in Activin signaling highlighted in pink and comparison of 3D
vs 2D in L’Oréal study and 3D vs 2D in Higgins study with genes involved in Activin signaling pathway highlighted in pink.
In L’Oréal study, culture in 3D structure regulated INHBB and FST in a way that might increase the biodisponibility of activin B in the extracellular space and participate in the
hair inductive capacity of DP cells by initiating the Activin signaling in epithelial cells. Of note, FST is an inhibitor of BMP2/4, the expression of which is increased during the
refractory period of telogen phase. Interestingly, the effect of 3D structure on INHBB and FST gene expressions is specific to L’Oréal compared to Higgins study.
CONCLUSION
In this study, the characterization of a new way of developing DP cells
spheroids generated by centrifugation shows that switching growth
in 2D to 3D represses effectors of cell cycle and myofibroblast differentiation which are initiated by growth of cells in 2D. In addition,
potent angiogenic factors are induced by growth of dermal papilla
cells in 3D. Examining the expression of components of signaling
pathways key to hair morphogenesis and hair follicle growth, we
found also that DP cell spheroids obtained by centrifugation tends
to activate WNT signaling, BMP signaling and NOTCH signaling within DP cells. However, genes involved in Activin signaling pathway
are specific to L’Oréal study compared to Higgins’ study.
We conclude that despite the different processes for aggregation of DP cells, the centrifugation (L’Oréal) and
hanging drop (Higgins) techniques are well correlated, as indicated by the correlation coefficient between the
studies. These two techniques also result in similar fold changes in gene expression between DP cells cultured in 3D versus 2D. Thus, the centrifugation DP cell spheroid model may provide us with a high throughput
screening tool for identification of new anti-hair loss and pro-hair growth compounds.
Biography
Florence Berthelot is currently a research scientist at L’Oréal Research and Innovation since 2012. She is part of the Micro-tissue lab under the
direction of Thomas Pocard PhD, at Predictive Methods and Models Department where she centers her work on the development of in vitro model
of hair and the development of read-out in hair loss axis. She obtained her Master degree in 2011 at Pierre and Marie Curie University of Sciences
in Paris in the fields of bio-engineering and biotechnologies. She joined L’Oréal for her final year traineeship in hair stem cell field at Stem Cell lab
under the direction of Maryline Paris PhD, at Biological and Clinical Research Department.
The authors declare no conflict of interest