Patent Application
20180245038
The present invention relates generally to a method for generating somatic stem cells and to somatic stem cells generated by said method. The invention further related to a vector, a composition and a kit for use in said method, for use in regenerative medicine, tissue repair, ex-vivo or in vivo modeling of human diseases, such as cancer, liver failure, diabetes, neurological deficiencies.
Stem cells (SCs) display the capacity to renew themselves when they divide, and to generate a differentiated progeny. Somatic SCs operate in multiple adult organs for continuous tissue renewal or repair after injury. Yet, these cells are still mainly defined by operational definitions and cell surface markers rather than the molecular traits that govern their special status (Fuchs, E. & Chen, T. A matter of life and death: self-renewal in stem cells. EMBO reports 14, 39-48 (2013)). Unlimited availability of normal, somatic SCs will be critical for effective organ repopulation in regenerative medicine applications, to understand SC biology and for disease modeling in the Petri dish. These efforts are frustrated by the fact that SCs are rare and difficult to purify from native tissues or to expand ex vivo. A recent important step forward in this direction has been the description of culture systems allowing adult epithelial SCs of endodermal origin to expand and self-organize into “organoids” (Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190-1194 (2013)). Yet, these methods still require the isolation of native SCs as starting material.
Direct conversion of terminally differentiated cells back into their corresponding tissue-specific SCs may represent an attractive alternative to obtain somatic SCs. Indeed, several reports have recently highlighted a surprising plasticity in somatic cell fates, as differentiated cells can return to a SC status under special conditions, such as tissue damage (Blanpain, C. & Fuchs, E. Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration. Science 344, 1242281 (2014); and Tetteh, P. W., Farin, H. F. & Clevers, H. Plasticity within stem cell hierarchies in mammalian epithelia. Trends in cell biology (2014)). However, the identity of the factors able to control the somatic SC status remains poorly understood, limiting the exploitation of such plasticity.
YAP (Yes-associated protein) and its paralog TAZ (transcriptional co-activator with PDZ-binding motif) are the main downstream effectors of the Hippo signaling pathway. This pathway is an evolutionally conserved signal cascade, which plays pivotal roles in organ size control and tumorigenesis from Drosophila to mammals (Guo, L and Teng, L, Int J Oncol. 2015 April; 46(4):1444-52.).
Possible roles of these pathways in direct conversion of terminally differentiated cells back into their corresponding tissue-specific SCs still remain elusive.
Evaluation of methods for generating somatic stem cells and searching for factors involved in the underlying molecular mechanisms continue.
The present invention provides a method for generating somatic stem cells and a somatic stem cell obtained by said method. The present invention further provides a vector, a composition and a kit for use in the method of the invention.
In one aspect, the present invention provides a method for generating somatic stem cells, comprising the steps of:
In some embodiments, the expression of the YAP/TAZ protein and/or the functional fragment of the YAP/TAZ and/or the activated version of the YAP/TAZ protein, or derivatives thereof in the at least one differentiated cell or committed progenitor or partially differentiated cell may be increased transiently. This improves the security of the invention, since the induced over expression of YAP/TAZ protein may be stopped once the somatic stem cell has been generated.
In some embodiments, said YAP/TAZ protein may be endogenous.
The activity of endogenous YAP/TAZ protein may be increased by influencing a biological activity of the endogenous YAP/TAZ protein, and/or by influencing a cellular stability of the endogenous YAP/TAZ protein, and/or by a influencing a cellular localization of the endogenous YAP/TAZ protein.
According to one embodiment, this may be done by applying to the at least one differentiated cell or committed progenitor or partially differentiated cell a composition comprising a substance for influencing the biological activity of the endogenous YAP/TAZ protein, and/or for influencing a cellular stability of the endogenous YAP/TAZ protein, and/or for influencing a cellular localization of the endogenous YAP/TAZ protein.
In accordance with one embodiment, the method may comprise the step of transfecting the at least one differentiated cell or committed progenitor or partially differentiated cell of step a) with a vector comprising a nucleotide sequence coding for a protein which induces the increased expression or activity of the endogenous YAP/TAZ protein.
According to some embodiments of the present invention, the increased expression of the YAP/TAZ protein and/or the functional fragment and or/said activated version, and/or derivatives thereof in the at least one differentiated cell or committed progenitor or partially differentiated cell may be ectopic. The method may then further comprise the step of transfecting the at least one differentiated cell of step a) with a vector comprising a nucleotide sequence coding for a wild-type YAP protein and/or a nucleotide sequence coding for a wild-type TAZ protein and/or a nucleotide sequence coding for a functional fragment of the wild-type YAP protein and/or the wild-type TAZ protein, and/or a nucleotide sequence coding for the activated version, and/or derivatives thereof.
The transfection of the at least one differentiated cell may be performed using a lentiviral vector. This allows for infection of non-dividing cells. Further, the vector can be integrated into the genome of the differentiated cell.
In accordance with one embodiment of the present invention, expression of the wild-type YAP/TAZ protein and/or the functional fragment of the YAP/TAZ protein and/or the activated version of the YAP/TAZ protein, and/or derivatives thereof is under the control of an inducible promoter. An example for such inducible promoter is a doxycyclin-inducible promoter. Transient expression may thereby be provided by the use of self-inactivating lentiviral vectors (in which the transgene may be deleted from the receiving cell genome) or by adenoviral vectors (that never integrate in the host genome) in order to improve the security of the method.
In the above method, the starting cell can be any mammalian cell, including, but not limited to, terminally differentiated cells. In some embodiments, the cell is a human cell, mouse cell, or rat cell. Examples of differentiated cells include, e.g., differentiated mammary gland cells, differentiated neural cells and differentiated pancreatic cells. The cell may be a terminal differentiated cell, a committed progenitor or a partially differentiated cell or a cell with dual stem-differentiated traits.
According to one embodiment, the step of generating a somatic stem cell comprises verifying at least one characteristic typical for somatic stem cells. For example, morphological characteristics of the cells may be used to check whether somatic stem cells have been generated. Alternatively or additionally, on a molecular level, it may be tested whether typical SC markers are detectable on the cell after executing step b) of the above method.
According to one embodiment, in order to verify the generation of a somatic stem cell, self renewal potential of the cell may be tested.
Alternatively or additionally, if the differentiated cells are differentiated mammary gland cells, the ability to self organize into mammary tissue like structures may be tested.
Further, multilineage differentiation ability of the cells may be tested in order to verify the generation of a somatic stem cell.
According to one embodiment, endogenous YAP/TAZ expression may be measured in the at least one differentiated cell or committed progenitor or partially differentiated cell after having stopped the induced increased expression of the ectopic YAP protein, and/or the TAZ protein, and/or the functional fragment of the YAP and/or the TAZ protein, and/or an activated version of the YAP and/or the TAZ protein, or derivatives thereof, in the at least one differentiated cell according to step b). Reactivation of endogenous YAP/TAZ expression may indicate the generation of somatic stem cells.
According to one embodiment, endogenous YAP/TAZ expression may be measured in the at least one differentiated cell or committed progenitor or partially differentiated cell after having stopped influencing a biological activity, a cellular stability or a cellular localization of an endogenous YAP/TAZ protein. Reactivation of endogenous YAP/TAZ expression after suspension of external activation may indicate the generation of somatic stem cells.
According to one embodiment, the step of generating a somatic stem cell comprises verifying the loss of expression of terminal differentiation markers of the cell after implementing step b) of the above method. Further, expression of typical SC markers may be measured.
Methods suitable to determine whether expression of YAP/TAZ or their biologically active derivative has reprogrammed a somatic cell into a stem cell include expression studies by means of polyacrylamide gel electrophoresis and related blotting techniques such as western blot paired with chromogenic or fluorescence and luminescence-based detection procedures; it also include immunofluorescence in cellular specimens aimed to determine acquired expression of genes typical of somatic SCs of a given tissue. Gene expression (i.e. downregulation of differentiated markers and upregulation of SC-markers) may be demonstrated by in situ hybridization and PCR-based procedure such as qPCR, RT-PCR, qRT-PCR, RT-qPCR, Light Cycler®, TaqMan® Platform and Assays, Northern blot, dot blot, microarrays, next generation sequencing (VanGuilder, Biotechniques (2008), 44: 619-26; Elvidge, Pharmacogenomics (2006), 7: 123-134; Metzker, Nat Rev Genet (2010), 11: 31-46). The corresponding experimental conditions are also established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (2001); Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Higgins and Hames (Eds.) “Nucleic acid hybridization, a practical approach” IRL Press Oxford, Washington D.C., (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art.
In addition to gene expression, the acquisition of a somatic SC fate can be measured by functional assays, in particular the acquisition of proliferative properties and ability of the induced/reprogrammed cell to be serially passaged and expanded, while retaining the ability to generate a differentiated progeny. Somatic SC acquisition can be also validated by the ability to regenerate tissues in animal models.
In another aspect, the present invention provides a somatic stem cell, obtained by anyone of the methods described above.
The induced somatic stem cell according to the present invention may be used in a regenerative medicine application. For example, the somatic stem cells may be used for generating tissues for transplantation. The somatic stem cells may be used to repair or replace tissue or organ function lost due to age, disease, organ damage, or congenital defects. The induced somatic stem cells may then be used to generate cells and tissue ex-vivo, to correct genetic defects, to expand or generate de novo stem cells in vivo, including self-propagating cells with augmented properties in comparison with natural/endogenous stem cells.
In a further aspect of the present invention, it is provided a vector comprising a nucleotide sequence coding for a wild-type YAP protein, and/or a nucleotide sequence coding for a wild-type TAZ protein, and/or a nucleotide sequence coding for a functional fragment of said YAP and/or said TAZ protein, and/or a nucleotide sequence coding for an activated version of said YAP and/or said TAZ protein, and/or a nucleotide sequence coding for a protein which induces an increased expression or activity of an endogenous YAP/TAZ protein, or derivatives thereof, wherein the transcription of said nucleotide sequence is under the control of an inducible promoter, for use in any one of the methods according to the present invention.
According to one embodiment, the nucleotide sequence may comprise anyone of the sequences Seq ID No. 1, Seq ID No. 2, Seq ID No. 3, or Seq ID No 4.
The vector may further comprise the nucleotide sequence according to Seq ID No. 5.
In a further aspect of the present invention, it is provided a composition comprising a substance for influencing a biological activity of an endogenous YAP/TAZ protein, and/or for influencing a cellular stability of said endogenous YAP/TAZ protein, and/or for influencing a cellular localization of said endogenous YAP/TAZ protein, for use in any one of the methods according to the present invention.
In a further aspect of the present invention, it is provided a kit, comprising a vector according to the present invention and/or comprising a composition in accordance with the present invention.
According to one embodiment, the kit may include a vector and/or a composition being prepared to be administered orally, rectally, by injection, inhalation, or topically.
k-l show mammary gland reconstitution generated by single-cell derived yMaSC organoids in a impregnated female. k: whole-mount images (left, native GFP fluorescence; right, hematoxylin staining). l: histological section. Note that upon gestation and lactation, the mammary gland is constituted by alveoli filled with milk.
a-b show representative images of a pancreatic duct fragment growing in pancreatic organoid medium at the indicated times, and after four passages in fresh Matrigel(b). Pictures are representative of three independent experiments performed with four technical replicates. Scale bars in a and b, 290 μm;
b-c refers to
f-g show serial images of a whole acinus derived from R26-rtTA; tetO-YAPS127A growing as cyst-like organoid at the indicated time points after Doxycycline addition (f) and after one or four passages in fresh Matrigel in the absence of Doxycycline (g). Scale bars, 70 μm in f; 290 μm in g; and
f shows qRT-PCRs for the indicated exocrine and Ductal/progenitor markers in fresh pancreatic acini, yDucts and Ducts. Data are normalized to 18SrRNA expression and are referred to Acini for exocrine differentiation markers, and to Ducts for Ductal/progenitor genes (each set to 1). Results are representative of four independent experiments performed in triplicate. Data are presented as mean+s.d.; and
g shows a representative immunofluorescences for the ductal marker K19 and the exocrine marker CPA1 before (day 0) and after yDuct differentiation (day 8). Similar results were obtained with organoids from normal ducts (not shown). Scale bar: 17 μm.
In one aspect, the present invention provides a method for generating somatic stem cells, comprising the steps of:
While induction of one of the increased expression or activity of a YAP protein, or a TAZ protein, or a functional fragment of the YAP or the TAZ protein, or an activated version of the YAP or the TAZ protein, or derivatives thereof, is sufficient to generate a somatic stem cell out of said differentiated cell, committed progenitor or partially differentiated cell, a combination of induced increase of expression of multiple of the proteins is also contemplated.
In some embodiments, the method comprises inducing the increased expression of the YAP protein, and/or the TAZ protein, and/or the functional fragment of the YAP and/or the TAZ protein, and/or the activated version of the YAP and/or the TAZ protein, or derivatives thereof in at least one differentiated cell or committed progenitor or partially differentiated cell (starting cell) transiently.
In some embodiments, the expression of the YAP protein, and/or the TAZ protein, and/or the functional fragment of the YAP and/or the TAZ protein, and/or the activated version of the YAP and/or the TAZ protein, or derivatives thereof in said at least one differentiated cell or committed progenitor or partially differentiated cell is increased transiently for a time sufficient for inducing the generation of a somatic stem cell out of the starting cell.
Once the induction of the generation of the somatic stem cell out of the starting cell has been initiated by the transient increase of expression of the YAP protein, and/or the TAZ protein, and/or the functional fragment of the YAP and/or the TAZ protein, and/or the activated version of the YAP and/or the TAZ protein, or derivatives thereof, the induced transient increase may be reduced and/or terminated.
Whilst not being bound by theory, it is thought that induced transient increase of expression of the YAP protein, and/or the TAZ protein, and/or the functional fragment of the YAP and/or the TAZ protein, and/or the activated version of the YAP and/or the TAZ protein, or derivatives thereof amongst other functions leading to the generation of a somatic stem cell out of the starting cell, initiates an expression of endogenous YAP/TAZ which is sufficient for maintaining stem cell properties in the generated somatic stem cell.
Transient induction of expression may thus be advantageously used in the method according to the present invention in order to improve security of the method. For example, obtained somatic stem cells may be used with reduced risk for adverse effects in regenerative medicine applications.
According to one embodiment, increased expression or increased activity of at least one endogenous YAP/TAZ protein in the cell is induced in the starting cell. This may be done by applying to the at least one differentiated cell, or committed progenitor, or partially differentiated cell a composition comprising a substance for influencing the biological activity of the endogenous YAP/TAZ protein, and/or for influencing a cellular stability of the endogenous YAP/TAZ protein, and/or for influencing a cellular localization of the endogenous YAP/TAZ protein. For example, inhibitors of endogenous expression of YAP/TAZ proteins in the starting cell may be blocked by the substance, or activation pathways for increase of expression of the endogenous YAP/TAZ proteins in the at least one differentiated cell may be activated by the substance.
The substance may activate a biological activity of the endogenous YAP/TAZ protein, by modulating e.g. a conformation and/or a modification of the endogenous YAP/TAZ protein. The substance may modulate the cellular localization of endogenous YAP/TAZ protein or increase the stability of endogenous YAP/TAZ protein. For example, the endogenous YAP/TAZ protein may be protected from degradation/digestion from cellular proteins. For example, the biological activity of the endogenous YAP/TAZ protein being influenced by the substance may be transcriptional activity of the endogenous YAP/TAZ protein. It is also contemplated that the substance modulates a histone modification for inducing increased expression of the endogenous YAP/TAZ protein. Of course, other generally known ways to induce an increase in gene expression may also be used by the substance for influencing the biological activity of the endogenous YAP/TAZ protein.
In a preferred embodiment, the increased expression and/or increased activity of the at least one endogenous YAP/TAZ protein in the cell is induced transiently.
According to one embodiment, in combination with or alternative to applying a substance to the starting cell in order to activate a biological activity of endogenous YAP/TAZ protein, the starting cell may be transfected with a vector comprising a nucleotide sequence coding for a protein which induces an increased expression or a biological activity of said endogenous YAP/TAZ protein.
The biological activity of endogenous YAP/TAZ is understood to be a biological activity which leads to the generation of somatic stem cells out of the at least one differentiated cell or committed progenitor or partially differentiated cell in accordance with the method of the present invention.
In one embodiment, the increased expression of said YAP/TAZ protein and/or said functional fragment and/or said activated version in said at least one differentiated cell is ectopic.
In a preferred embodiment, said at least one differentiated cell of step a) is transfected with a vector comprising a nucleotide sequence coding for a wild-type YAP protein and/or a nucleotide sequence coding for a wild-type TAZ protein and/or a nucleotide sequence coding for a functional fragment of said wild-type YAP protein and/or said wild-type TAZ protein, and/or a nucleotide sequence coding for said activated version, or derivatives thereof.
Preferably, a nucleotide sequence coding for a wild-type YAP protein is used as set forth in Seq. ID No 1.
Preferably, a nucleotide sequence coding for a wild-type TAZ protein is used as set forth in Seq. ID No 2.
Preferably, a nucleotide sequence coding for an activated version of the YAP protein is used as set forth in Seq. ID No 3.
Preferably, a nucleotide sequence coding for an activated version of the TAZ protein is used as set forth in Seq. ID No 4.
In yet other specific embodiments, the present invention provides a vector comprising a nucleotide sequence having at least 70%, 80%, 90%, or 95% identity to at least 60 nucleotides of the sequences set forth in SEQ ID No's 1, 2, 3 or 4.
The transfection of said at least one differentiated cell may be performed using a lentiviral vector. Further, the expression of said wild-type YAP/TAZ protein and/or said functional fragment and or/said activated version may be under the control of an inducible promoter. For example, said inducible promoter may be a doxycyclin-inducible promoter. Such promoter has been described e.g. in U.S. Pat. Nos. 5,814,618, 7,541,446, and 8,383,364. However, other inducible promoter-systems which are generally known in the art are also contemplated. Use of this vector system allows for easy controlling of the transient induction period for increased expression of said wild-type YAP/TAZ protein and/or said functional fragment and or/said activated version.
In a preferred embodiment of the present application, a doxycyclin inducible promoter is used according to a nucleotide sequence as set forth in Seq ID No 5. Such tetO promoter system has been described e.g. by Bujard, Hermann and M. Gossen (“Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters; (Proc. Natl. Acad. Sci. U.S.A. 89 (12): 5547-51).
If the somatic stem cell has been generated by carrying out step b) of the method according to the present invention for a sufficient time, the transfected nucleotide sequence coding for the wild-type YAP protein and/or the nucleotide sequence coding for the wild-type TAZ protein and/or the nucleotide sequence coding for the functional fragment of said wild-type YAP protein and/or said wild-type TAZ protein, and/or the nucleotide sequence coding for said activated version or derivatives thereof may be removed from the generated somatic stem cell. Such removal of the transfected nucleotide sequence may be carried out according to the standard methods known in the art, depending on the vector system used for transfection.
As used herein, the term “sufficient time” shall mean a period sufficiently long to reprogram the differentiated cell by the transient induction of increased expression of the YAP/TAZ proteins disclosed herein.
In some embodiments, the term “sufficient time” shall mean at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, or at least 30 days.
In some embodiments, the term “sufficient time” ranges from 1 day to about 180 days, e.g., from about 1 day to about 2 days, from about 1 day to about 7 days, from about 1 day to about 14 days, from about 1 day to about 21 days, from about 1 day to about 30 days, from about 1 day to about 45 days, from about 1 day to about 60 days, from about 1 day to about 90 days, from about 1 day to about 120 days, from about 1 day to about 150 days, or from about 1 day to about 180 days.
In some embodiments, the term “sufficient time” ranges from 2 days to about 180 days, e.g., from about 2 days to about 7 days, from about 2 days to about 14 days, from about 2 days to about 21 days, from about 2 days to about 30 days, from about 2 days to about 45 days, from about 2 days to about 60 days, from about 2 days to about 90 days, from about 2 days to about 120 days, from about 2 days to about 150 days, or from about 2 days to about 180 days.
As further used herein, the term vector is understood to mean any DNA molecule that can be used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed.
As further used herein, the term functional fragment is understood to mean a truncated and/or incomplete form of a YAP/TAZ protein which still harbors its functional activity to induce de novo generation of a somatic stem cell out of a more differentiated cell.
In another aspect of the present invention, it is provided a somatic stem cell obtained by the method according to the present invention. The induced somatic stem cell according to the present invention may be used in a regenerative medicine application. For example, the somatic stem cells may be used for generating tissues for transplantation. The somatic stem cells may be used to repair or replace tissue or organ function lost due to age, disease, organ damage, or congenital defects. The induced somatic stem cells may then be used to generate cells and tissue ex-vivo, to correct genetic defects, to expand or generate de novo stem cells in vivo, including self-propagating cells with augmented properties in comparison with natural/endogenous stem cells.
In another aspect of the present invention, a vector for use in the method of the present application is provided; the vector comprising a nucleotide sequence coding for a wild-type YAP protein, and/or a nucleotide sequence coding for a wild-type TAZ protein, and/or a nucleotide sequence coding for a functional fragment of said YAP and/or said TAZ protein, and/or a nucleotide sequence coding for an activated version of said YAP and/or said TAZ protein, wherein the transcription of said nucleotide sequence is under the control of an inducible promoter.
In accordance with the present invention, the inhibitor (i.e. in case of a nucleic acid inhibitor) of the polynucleotide to be inhibited in context of the present invention may be cloned into a vector. The term “vector” as used herein particularly refers to plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. In a preferred embodiment, these vectors are suitable for the transformation of cells, like fungal cells, cells of microorganisms such as yeast or prokaryotic cells. In a particularly preferred embodiment, such vectors are suitable for stable transformation of bacterial cells, for example to transcribe the polynucleotide of the present invention.
Accordingly, in one aspect of the invention, the vector as provided is an expression vector. Generally, expression vectors have been widely described in the literature. As a rule, they may not only contain a selection marker gene and a replication-origin ensuring replication in the host selected, but also a promoter, and in most cases a termination signal for transcription. Between the promoter and the termination signal there is preferably at least one restriction site or a polylinker which enables the insertion of a nucleic acid sequence/molecule desired to be expressed.
It is to be understood that when the vector provided herein is generated by taking advantage of an expression vector known in the prior art that already comprises a promoter suitable to be employed in context of this invention, for example expression of an inhibitor (i.e. in case of a nucleic acid inhibitor) of a polynucleotide as described hereinabove, the nucleic acid construct is inserted into that vector in a manner the resulting vector comprises only one promoter suitable to be employed in context of this invention. The skilled person knows how such insertion can be put into practice. For example, the promoter can be excised either from the nucleic acid construct or from the expression vector prior to ligation.
As a non-limiting example, a vector comprising a nucleotide sequence coding for a wild-type YAP protein, and/or a nucleotide sequence coding for a wild-type TAZ protein, and/or a nucleotide sequence coding for a functional fragment of said YAP and/or said TAZ protein, and/or a nucleotide sequence coding for an activated version of said YAP and/or said TAZ protein, and/or a nucleotide sequence coding for a protein which induces an increased expression or activity of an endogenous YAP/TAZ protein, or derivatives thereof, is cloned is an adenoviral, adeno-associated viral (AAV), retroviral, or nonviral minicircle-vector. Further examples of vectors suitable to comprise an inhibitor (i.e. in case of a nucleic acid inhibitor) of a polynucleotide to be inhibited in order to induce increased expression of an endogenous YAP/TAZ protein in context of the present invention to form the vector described herein are known in the art.
In an additional embodiment, the coding nucleic acid sequence of an inducer of YAP/TAZ in context of the present invention and/or the vector into which the polynucleotide described herein is cloned may be transduced, transformed or transfected or otherwise introduced into a host cell. For example, the host cell is a eukaryotic or a prokaryotic cell, for example, a bacterial cell. As a non-limiting example, the host cell is preferably a mammalian cell. The host cell described herein is intended to be particularly useful for generating the inhibitor of a polynucleotide to be inhibited in context of the present invention. An inducer of YAP/TAZ is intended as a polynucleotide sequence able to activate YAP/TAZ nuclear localization and transcriptional activation (as determined by luciferase assays and activation or YAP/TAZ direct target genes such as CTGF) Dupont et al., Nature 2011).
An overview of examples of different corresponding expression systems to be used for generating the host cell described herein is for instance contained in Methods in Enzymology 153 (1987), 385-516, in Bitter (Methods in Enzymology 153 (1987), 516-544), in Sawers (Applied Microbiology and Biotechnology 46 (1996), 1-9), Billman-Jacobe (Current Opinion in Biotechnology 7 (1996), 500-4), Hockney (Trends in Biotechnology 12 (1994), 456-463), and in Griffiths (Methods in Molecular Biology 75 (1997), 427-440). The transformation or genetically engineering of the host cell with a polynucleotide to be inhibited in context of the present invention or vector described herein can be carried out by standard methods, as for instance described in Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, N.Y., USA; Methods in Yeast Genetics, A Laboratory Course Manual, Cold Spring Harbor Laboratory Press, 1990.
The following examples illustrate, rather than limit, embodiments of the present invention.
The mammary gland represents a classic model system for the study of epithelial SCs and tissue regeneration. Remarkably, implantation of mammary gland SCs (MaSCs) into the mammary fat pad is sufficient to regenerate an entire ductal tree, with MaSCs contributing to both the luminal and myoepithelial lineages.
To address whether expression of YAP/TAZ may bestow stemness characteristics also to normal mammary cells, freshly dissected, lineage negative (Lin-) and EpCAM positive mammary epithelial cells were FACS-purified using the CD61 and CD49f cell surface antigen markers (
To investigate whether ectopic expression of YAP or TAZ in LD cells could impart MaSC-like properties, FACS-purified LD cells were plated on collagen-coated dishes and transduced with doxycycline-inducible lentiviral vectors encoding for wild-type (wt) YAP, or the activated versions of YAP and TAZ (i.e., YAP5SA or TAZ4SA, lacking inhibitory phosphorylation sites) (see diagram in
It has been examined whether increased YAP/TAZ expression may convert luminal cells to a MaSC-like state. First, it has been addressed whether YAP/TAZ expression endowed self-renewal potential, a fundamental SC trait that can be assayed in vitro by the ability to serially passage mammary colonies. YAP/TAZ-induced colonies, similarly to those generated from MaSCs, could form additional generations of colonies after single cell dissociation. Notably, the colony-forming efficiency after passaging was comparable in presence and absence of doxycycline, that is, irrespective of ectopic YAP/TAZ expression (Extended Data
To verify whether the switch from LD to a MaSC-like state could be recapitulated at the single cell level, individual LD cells were seeded in 96-well plates (visually verified) and induced to express YAP. By monitoring the resulting outgrowths, it has been found that 18% of these individual cells formed solid colonies (
It was then established if yMaSCs truly represented mammary SCs, as determined by additional cardinal properties of SCs, such as the ability to self-organize in vitro into mammary tissue-like structures, to differentiate along distinct lineages, and to regenerate a mammary tree in vivo after injection into a cleared mammary fat pad. For this, a long-term culture system has been established that allows yMaSCs to form mammary-gland like structures in vitro. MaSC- and yMaSC-derived colonies were transferred and embedded into 100% Matrigel, and overlaid with “organoid” medium containing EGF, bFGF, Noggin, B27, and R-Spondin112 in absence of doxycycline. Under these conditions, colonies underwent extensive budding and, by 2 weeks, grew into large epithelial organoids (
To further validate the notion that YAP expression converts differentiated cells to a SC fate genetic lineage-tracing experiments have been carried out using LD cells irreversibly labeled with YFP purified from K8-CreERT2; R26-LSL-YFP mice (
To characterize at the molecular level the similarities between yMaSCs to their natural counterpart, we compared FACS-purified SCs (EpCAMlowCD49fhighCD61+) from the mammary gland and yMaSC-induced organoids. Purified LD cells were used as control. As shown in
To reinforce the notion that MaSCs and yMaSCs are similar, the gene-expression profiles of their respective organoid cultures, and of LD cells have been compared (
Next, it has been tested whether yMaSCs displayed mammary gland reconstituting activity. For this, FACS-purified LD cells were transduced with vectors encoding for EGFP and inducible wild-type YAP. Cells were treated with doxycycline for 7 days and then transplanted (103-104 cells) into the cleared mammary fat pad of NOD-SCID mice, kept in a doxycycline-free diet for 10 weeks. Strikingly, cells that had experienced transient expression of wild-type YAP had also acquired the ability to regenerate the mammary gland (25%, n=16) (
To explore the reconstituting potential of a single yMaSC, in the cleared fat pads single-cell derived organoids have been injected, and it was found that these were also able to regenerate the mammary gland (33%, n=6). Notably, when these mice were impregnated, reconstituted mammary glands generated a dense ductal system ending in clusters of milk-secreting alveoli, indicating that yMaSCs retain full differentiation potential in vivo (
Next, it was checked whether SC-generation by ectopic YAP expression was specific for mammary epithelial cells, or rather represented a more general principle. This question has been addressed in neurons, a cell type considered a classic example of terminal differentiation.
Neurons were prepared by dissociating the hippocampus or cortex of late mouse embryos (E19), and selected for post-mitotic neurons by culturing primary cells in neuronal-differentiation medium containing AraC for 4-7 days (Han, X. J. et al. CaM kinase I alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology. The Journal of cell biology 182, 573-585 (2008)). This procedure eliminates proliferating cells, resulting in a population of mature post-mitotic neurons (>95%) displaying multiple neurites and expressing βIII-Tubulin (TuJ1), NeuN and other typical neuronal markers (see below and
It has also been tested whether ectopic expression of YAP/TAZ in neurons was sufficient to convert them into NSCs. For this, primary cells were infected with lentiviral vectors encoding for rtTA and inducible wild-type YAP (see Methods). After AraC, neurons were shifted to NSC medium in the presence of doxycycline (see experimental outline in
To validate that the NSC-like cells were indeed derived from terminally differentiated neurons, YAP-induced reprogramming in genetically lineage-traced neurons has been repeated. For this, mice carrying the established neuronal driver Thy1-Cre20 and the R26-LSL-LacZ reporter have been used (see scheme in
Next, yNSCs have been characterized by immunofluorescence and marker gene expression. As shown in
In order to characterize to what extent YAP triggers neuronal conversion to a bona-fide NSC-status, the transcriptome of parental neurons, yNSCs and control NSCs have been compared. As shown in
Neural SCs are defined as tripotent, as defined by their ability to differentiate in astrocytes, neurons and oligodendrocytes. The developmental potential of yNSCs was thus examined and compared to NSCs. yNSCs plated on fibronectin and treated with BMP4 and LIF22 completely switched to a typical astrocyte morphology, also expressing high levels of GFAP (
Pancreatic progenitors purified from the pancreatic duct have been recently shown to be expandable in vitro as organoids (Huch, M. et al. Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. The EMBO journal 32, 2708-2721 (2013)) (
Aiming to exploit this fate plasticity, it was then checked whether YAP expression could convert explanted primary acinar cells, normally void of endogenous YAP/TAZ, into ductal progenitors in vitro. To this end, pancreatic acini from R26-rtTA; tetOYAPS127A adult mice were isolated and dissociated to obtain a cell preparation highly enriched in exocrine cells (>400 fold, see Methods). Cells were plated in 100% Matrigel and added of doxycycline in pancreas organoid medium (see scheme in
As an alternative strategy (avoiding primary acinar cells the harsh treatment of single cell dissociation with trypsin), whole pancreatic acini explanted from R26-rtTA; tetOYAPS127A have been embedded in collagen and cultured in low serum, that is, conditions that have been shown to preserve acinar cell identity ex-vivo (Means, A. L. et al. Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 132, 3767-3776 (2005)) (see also experimental outline in
To validate that yDucts were indeed derived from differentiated exocrine acinar cells, genetic lineage tracing experiments were carried out using Ptf1a-CreERTM; R26-LSL-rtTA-IRES-EGFP; tetO-YAP S127A mice. It has been previously shown that, in the Ptf1a-CreERTM; R26-LSL-rtTA-IRES-EGFP background, tamoxifen treatment of adult mice causes irreversible genetic tracing of pancreatic acinar cells exclusively (Pan, F. C. et al. Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration. Development 140, 751-764 (2013)). One-week post-tamoxifen, pancreata were explanted to prepare whole acini or single-cell dissociated acinar cells, that were cultured as above (see experimental outline in
In section, organoids appeared as epithelial monolayers surrounding a central cavity (
The present invention shows for the first time that expression of a single factor, YAP, into terminally differentiated cells explanted from different tissues efficiently creates cells with functional and molecular attributes of their corresponding tissue-specific SCs, that can be expanded ex-vivo as organoid cultures. The ySC state can be transmitted through cell generations without need of continuous expression of ectopic YAP/TAZ, indicating that a transient activation of ectopic YAP or TAZ is sufficient to induce a heritable self-renewing state.
According to the present invention, YAP/TAZ proteins are presented at the centerpiece of the somatic SC state whenever natural, pathological or ex-vivo conditions demand de novo generation and expansion of resident or facultative SCs.
The generation of autologous induced-SCs from various tissues by YAP/TAZ according to the present invention also holds the possibility to investigate somatic stemness or to expand rare cells, particularly in conditions in which aging or diseases have exhausted the endogenous SC pool. Finally, the present invention also raise the prospects to boost the body's regenerative capacity by sustaining YAP/TAZ expression at injury sites or as transplanted “super-SCs” able to produce new and more functional tissues than regular SCs.
Doxycycline hyclate, fibronectin, collagen I, heparin, insulin, dexamethasone, SBTI (Soybean Trypsin Inhibitor), gastrin, N-acethylcysteine, nicotinamide, T3 (Triiodo-L-Thyronine), tamoxifen and 4-OH-tamoxifen were from Sigma. Murine EGF, murine bFGF, human FGF10, human Noggin, human IGF, murine prolactin and BMP4 were from Peprotech. N2, B27, BPE and ITS-X (Insulin-Transferrin-Selenium-Ethanolamine) supplements were from Life Technologies. R-Spondin1 was from Sino Biological. Matrigel was from BD Biosciences (Corning). Rat tail collagen type I was from Cultrex. GFP- and Cre-expressing adenoviruses were from University of Iowa, Gene Transfer Vector Core. For inducible expression of YAP and TAZ, cDNA for siRNA-insensitive Flag-hYAP1 wt, S94A (TEAD-binding mutant, Zhao, B. et al. TEAD mediates YAP-dependent gene induction and growth control. Genes & development 22, 1962-1971 (2008))) and 5SA (LATS-mutant sites)(Aragona, M. et al. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154, 1047-1059 (2013)) and for Flag-mTAZ4SA (Azzolin, L. et al. Role of TAZ as mediator of Wnt signaling; Cell 151, 1443-1456 (2012)) were subcloned in FUW-tetO-MCS, obtained by substituting the Oct4 sequence in FUW-tetO-hOct4 (Addgene #20726 (Hockemeyer, D. et al. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency; Cell Stem Cell 3, 346-353 (2008)) with a new multiple cloning site (MCS). This generated the FUW-tetO-wtYAP, FUW-tetO-YAPS94A, FUW-tetO-YAP5SA, FUW-tetO-TAZ4SA used throughout this study. FUW-tetO-MCS (empty vector) or FUW-tetO-EGFP plasmids were used as controls, as previously indicated8. All available in Addgene as #.
For stable expression of GFP, we used pRRLSIN.cPPT.PGK-GFP.WPRE (gift of L. Naldini) lentiviral vector.
For Cre-excisable expression of rtTA, we used LV-CMV-rtTA-LoxP (see scheme Extended Data
For Cre-excisable lentiviral vector containing the tetO-Flag-hYAP wt cassette, we used LV-tetO-YAP wt-LoxP (see scheme
All constructs were confirmed by sequencing.
siRNA transfections were done with Lipofectamine RNAi-MAX (Life technologies) in antibiotics-free medium according to manufacturer instructions. Sequences of siRNAs targeting murine Yap and Taz are as previously described (zzolin, L. et al. YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response. Cell 158, 157-170 (2014)).
DNA transfections were done with TransitLT1 (Minis Bio) according to manufacturer instructions.
Lentiviral particles were prepared by transiently transfecting with TransIT-LT1 in Opti-MEM lentiviral vectors (10 micrograms/10 cm dishes) together with packaging vectors pMD2-VSVG (2.5 micrograms) and pPAX2 (7.5 micrograms) in HEK293T cells (checked routinely for absence of mycoplasma contaminations). Virus-producing HEK293T cells were cultured in DMEM (Life Technologies), supplemented with 10% FBS, glutamine and antibiotics. Supernatants were collected 48 hours post-transfection and lentiviral titer was determined using the QuickTiter Lentivirus Titer kit (lentivirus-associated HIV p24; Cell Biolabs) according to the manufacturer's protocol. The collected supernatant were filtered through 0.45 micrometers and directly stored at −20° C.; we did not concentrate viral supernatants. Each viral supernatant was used at a final titer of about 2-5 ng of p24/ml (see specifics below). In our hands, this typically corresponds to a simple 1:4 dilution of the each viral supernatant, in turn corresponding to a working final viral particle concentration of about 5×107 particles/ml. As determined by PCR of integrated lentiviral DNA of HEK293T transduced with pRRL-EGFP, this roughly corresponds to 5×105 transduction units (TU)/ml.
Primary Mammary Epithelial Cells (MECs) Isolation and Induction of yMaSCs
Primary MECs were isolated from the mammary glands of 8- to 12-week-old virgin C57BL/6J mice (unless otherwise specified), according to standard procedures. Mammary glands were minced and then digested with 6000 U/ml collagenase I (Life Technologies) and 2000 U/ml hyaluronidase (Sigma) in the DMEM/F12 (Life Technologies) at 37° C. for 1 hour with vigorous shaking. The digested samples were pipetted, spun down at 1500 rpm for 5 min, and incubated 3 min in 0.64% buffered NH4Cl (Sigma) in order to eliminate contaminating red blood cells. After washing with DMEM/F12+5% FBS, cells were plated for 1 hour at 37° C. in DMEM/F12+5% FBS: in this way, the majority of fibroblasts attached to the tissue culture plastic, whereas mammary epithelial populations did not and were therefore recovered in the supernatant. After washing in PBS/EDTA 0.02%, MECs were further digested with 0.25% trypsin (Life Technologies) for 5 min and 5 mg/ml dispase (Sigma) plus 100 μg/ml DNase I (Roche) for other 10 min. The digested cells were diluted in DMEM/F12+5% FBS and filtered through 40 μm cell strainers to obtain single cell suspensions cells and washed once in the same medium.
For separating various MEC subpopulations cells were stained for 30 min at 4° C. with antibodies against CD49f (PE-Cy5, cat. 551129, BD Biosciences), CD29 (PE-Cy7, cat. 102222, BioLegend), CD61 (PE, cat. 553347, BD Biosciences), EpCAM (FITC, cat. 118208, BioLegend) and lineage markers (APC mouse Lineage Antibody Cocktail, cat. 51-9003632, BD Biosciences) in DMEM/F12.
The stained cells were then resuspended in PBD/BSA 0.1% and sorted on a BD FACS Aria sorter (BD Biosciences) into luminal differentiated (LD) cells, luminal progenitor (LP) cells and mammary stem cells (MaSCs).
Primary sorted subpopulations from FACS were plated on collagen I-coated supports and cultured in 2D in mammary (MG) medium (DMEM/F12 supplemented with glutamine, antibiotics, 10 ng/ml murine EGF, 10 ng/ml murine bFGF, and 4 μg/ml heparin with 2% FBS).
For induction of yMaSCs, adherent luminal differentiated cells were transduced for 48 hours with FUW-tetO-YAP, or FUW-tetO-TAZ, in combination with rtTA-encoding lentiviruses. As a (negative) control, LD cells were transduced with FUW-tetO-EGFP (
For the experiment depicted in
After infection in 2D cultures and induction with doxycycline for 7 days, mammary cells were detached with trypsin and seeded at a density of 1,000 cells/well in 24-well ultralow attachment plates (Corning) in MG-colony medium (DMEM/F12 containing glutamine, antibiotics, 5% Matrigel, 5% FBS, 10 ng/ml murine EGF, 20 ng/ml murine bFGF, and 4 μg/ml heparin) containing doxycycline (2 μg/ml). Primary colonies were counted 14 days after seeding. To show the self-renewal capacity of yMaSCs independently of exogenous YAP/TAZ supply (i.e., independently of doxycycline administration), primary colonies were recovered from the MG-colony medium by collecting the samples and incubation in ice cold HBSS. Cells were dissociated and re-seeded in ultralow attachment plates in MG-colony medium without doxycycline for further passaging.
For mammary organoid formation, primary colonies were recovered from MG colony medium in cold HBSS and transferred in 100% Matrigel. After Matrigel formed a gel, MG organoid medium was added (Advanced DMEM/F12 supplemented with Hepes, GlutaMax, antibiotics, EGF, bFGF, heparin, noggin and R-Spondin1). Note that at this step we do not dissociate at single cell level the primary colonies but simply transfer them to organoid culture conditions. Also note that direct plating of MaSCs, LD control EGFP-infected, as well as YAP-infected cells, directly into organoid culture conditions did not result in any outgrowth, indicating that the intermediate step in colony culture conditions is required for organoid development. After few days, colonies started to form budding organoids. 2 weeks after seeding, organoids were removed from Matrigel as before, trypsin-dissociated and transferred to fresh Matrigel. Passages were performed in a 1:4-1:8 split ratio every 2 weeks for at least 9 months. For analysis, organoids were recovered from Matrigel as before, and either embedded in OCT medium (PolyFreeze, Sigma) to obtain frozen sections for immunofluorescence or processed for protein or RNA extraction. For α- and β-casein induction (
For induction of yMaSCs meant for in vivo injection (
Cell aliquots resuspended in 10 μl PBS/10% Matrigel were injected into the inguinal mammary fat pads of NOD-SCID mice (Charles River), which had been cleared of endogenous mammary epithelium at 3 weeks of age. Animals were then administered doxycycline in the drinking water for 2 weeks and then maintained without doxycycline for additional 8-10 weeks. Transplanted mammary fat pads were examined for gland reconstitution by whole-mount staining, GFP native fluorescence and immunofluorescence on sections from paraffin-embedded biopsies. Only the presence of GFP-positive branched ductal trees with lobules and/or terminal end buds was scored as positive reconstitution. For whole-mount analysis of mammary glands, freshly-explanted glands were fixed in PFA 4% (2 hours) and ethanol 70% (overnight). Glands were rehydrated, stained overnight with hematoxylin, subsequently dehydrated in graded ethanols, cleared by incubation in benzyl-alcohol/benzyl benzoate (1:2; Sigma) and imaged.
Primary Neuron Isolation and Induction of yNSCs
Neurons were prepared from hippocampi or cortices of E18-19 embryos as previously described (Han, X. J. et al. CaM kinase I alpha-induced phosphorylation of Drp1 regulates mitochondrial morphology. The Journal of cell biology 182, 573-585 (2008)). Briefly, hippocampi and cortices were dissected under the microscope in ice cold HBSS as quick as possible, incubated with 0.05% trypsin (Life Technologies) 15 min at 37° C. and, after trypsin blocking, resuspended in DMEM/10% FBS supplemented with 0.1 mg/ml DNase I (Roche), and mechanically dissociated. Cells were then plated on poly-L-lysine-coated plates in DMEM (Life technologies) supplemented with 10% FBS, glutamine and antibiotics for hippocampal neurons or in DMEM/Neurobasal (1:1) supplemented with 5% FBS, 1× B27, glutamine and antibiotics for cortical neurons (day 1). After 24 hours (day 2), medium was changed to fresh DMEM/Neurobasal (1:1) supplemented with 5% FBS, 1× B27, glutamine and antibiotics and, when specified, the next day (day 3) cells were infected with FUW-tetO-wtYAP and FUdeltaGW-rtTA viral supernatants. Negative controls were provided by neurons transduced with FUdeltaGW-rtTA alone or in combination with FUW-tetO-EGFP or FUW-tetO-MCS (empty vector). Viral supernatants were used at a final titer of about 4-5 ng of p24/ml for FUdeltaGW-rtTA, and 2 ng of p24/ml for all other viruses (see above the paragraph lentivirus preparation). After 24 hour (day 4), cells were incubated in Neurobasal medium supplemented with 1× B27, glutamine, antibiotics, and 5 μM Ara-C (cytosine β-D-arabinofuranoside; Sigma) for additional 7 days at the end of which well-differentiated, complex network-forming neurons are visible. To induce yNSCs formation, treated neurons were switched to NSC medium (DMEM/F12 supplemented with 1× N2, 20 ng/ml murine EGF, 20 ng/ml murine bFGF, glutamine, and antibiotics) and 2 μg/ml doxycycline for activating tetracycline-inducible gene expression. After 7 days, half of this medium was substituted with fresh NSC medium containing 4 μg/ml doxycycline. Sphere formation was evident upon YAP induction after 10-14 days of doxycycline treatment.
Spheres were gently transferred into a 15 ml-plastic tube and let sediment (typically 10-15 min). After discarding the supernatant, spheres were transferred to new Petri dishes with fresh NSC medium without doxycycline and let grow for 3-4 additional days. These neurospheres were then dissociated to single cells with TrypLE Express (Life Technologies), resuspended in NSC medium without doxycycline and transferred to a new dish; this step was repeated for every passage, as for normal NSCs.
For the experiment depicted in
For the experiment depicted in
For the experiment depicted in
For the experiment with excisable YAP vectors (
Neural stem cells (NSCs) were isolated as previously reported (Ray, J. & Gage, F. H. Differential properties of adult rat and mouse brain-derived neural stem/progenitor cells. Molecular and cellular neurosciences 31, 560-573 (2006)) from the telencephalon of C57BL/6J E18 embryos or from mice of the indicated genotype. Telencephalons were minced and digested in trypsin 0.05% for 10 min at 37° C. The cell suspension was treated with DNaseI (Roche) and washed. NSCs were cultured in DMEM/F12 supplemented with N2, 20 ng/ml murine EGF, 20 ng/ml murine bFGF, glutamine and antibiotics. For passages, neurospheres were dissociated into single cells with TrypLE Express (Life Technologies).
Prior to transfection with siRNA, yNSCs were plated on fibronectin coated-plate in NSC medium, to allow a 2D culture; the next day, cells were transfected with siRNA and after 24 hours, replated in ultra-low attachment plates to allow neurosphere formation. Neurospheres were counted after 7 days from plating.
For adenoviral infection of wild-type (wt) or double Yapfl/fl; Tazfl/fl NSCs (
For neuronal differentiation, NSCs or yNSCs were cultured over a thin Matrigel layer. Differentiation medium was Neurobasal supplemented with 1× B27, glutamine.
For astrocyte differentiation, NSCs or yNSCs were plated on fibronectin coated-plate in NSC medium, to allow a 2D culture. The next day, medium was changed to DMEM (Life Technologies) containing 25 ng/ml LIF, 25 ng/ml BMP4, glutamine, and antibiotics for 2 weeks.
For oligodendrocyte differentiation, NSCs or yNSCs were plated on fibronectin coated-plate in NSC medium, to allow a 2D culture. The next day, medium was changed to Neurobasal (Life Technologies) containing 1× B27, 500 ng/ml IGF, 30 ng/ml T3, glutamine, and antibiotics for 2 weeks.
P0 CD1 mice pups were used for cell transplantations. Pups were anesthetized by hypothermia (3 minutes) and fixed on ice-cold block during cell injection. Cells were resuspended in ice-cold HBSS (5×104 cells/μl) and injected into both hemispheres of neonatal mice with a 5 μl-volume Hamilton syringe (2 μl/injection). One month after the procedures, the grafted animals were perfused with PBS and 4% PFA, and the brains were excised and processed for immunofluorescence.
Pancreatic Acinar Cells Isolation and Induction of yDucts
Primary pancreatic acini were isolated from the pancreas of 6- to 9-week-old mice, according to standard procedures (Means, A. L. et al. Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 132, 3767-3776 (2005)). Digested tissue was filtered through a 100 μm nylon cell strainer. The quality of isolated acinar tissue was checked under the microscope. For culture of entire acini, explants were seeded in neutralized rat tail collagen type I (Cultrex)/acinar culture medium (1:1) (Means, A. L. et al. Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 132, 3767-3776 (2005)), overlaid with acinar culture medium (Waymouth's medium (Life Technologies) supplemented with 0.1% FBS (Life Technologies), 0.1% BSA, 0.2 mg/ml SBTI, 1×ITS-X (Life Technologies), 50 μg/ml BPE (Life Technologies), 1 μg/ml dexamethasone (Sigma), and antibiotics) once collagen formed a gel. For culture of isolated acinar cells, acini were further digested in 0.05% trypsin for 30 min at 37° C. to obtain a single cell suspension. Single acinar cells were plated in 100% Matrigel; once Matrigel formed a gel, cells were supplemented with pancreatic organoid medium (Advanced DMEM/F12 supplemented with 1× B27, 1.25 mM N-Acetylcysteine, 10 nM gastrin, 50 ng/ml murine EGF, 100 ng/ml human Noggin, 100 ng/ml human FGF10, 10 mM Nicotinamide, 1 μg/ml R-Spondin1 and antibiotics) supplemented with 0.2 mg/ml SBTI. To assess enrichment of acinar cells in our preparation, we compared RNA extracts from whole pancreas and our fresh acinar cell preparation for expression of exocrine cell markers, such as α-amylase, elastase and CPA1 (data not shown).
For induction of pancreatic organoids, entire acini or single acinar cells of the indicated genotypes cells were treated with 2 μg/ml doxycycline. Negative control cells were cultured in the same conditions in absence of doxycycline. Cells were treated with 2 μg/ml doxycycline for 7 days and organoid formation was morphologically followed. Organoids were then processed for further analyses.
For the experiment depicted in
Matrigel Culture of yDucts Organoids
To show the self-renewal capacity of pancreatic organoids independently of exogenous YAP supply (i.e., independently of doxycycline administration), organoids were recovered from Matrigel or collagen cultures, trypsinized to obtain a single cell suspension and re-seeded in 100% Matrigel covered with pancreatic organoid medium. For analysis, organoids were recovered from Matrigel as before and processed for immunofluorescence or for protein or RNA extraction.
For the differentiation experiments shown in
For culture of pancreatic duct-derived organoids, pancreatic ducts were isolated from the bulk of the pancreas as previously described25 with minor modifications. The whole pancreas of 6- to 9-week-old mice of the indicated genotypes was grossly minced and digested by collagenase/dispase dissociation: DMEM medium (Life Technologies) supplemented with collagenase type XI 0.012% (w/v) (Sigma), dispase 0.012% (w/v) (Life Technologies), 1% FBS (Life Technologies) and antibiotics at 37° C. for 1 hour. Isolated pancreatic duct fragments were hand-picked under a dissecting microscope, carefully washed in DMEM medium and embedded in 100% Matrigel. After Matrigel formed a gel, pancreatic organoid medium (Advanced DMEM/F12 supplemented with 1× B27, 1.25 mM N-Acetylcysteine, 10 nM gastrin, 50 ng/ml murine EGF, 100 ng/ml human Noggin, 100 ng/ml human FGF10, 10 mM Nicotinamide, 1 μg/ml R-Spondin1 and antibiotics) was added. Ductal fragments rapidly expanded to form cyst-like organoids within 5 days. Organoids were removed from Matrigel by incubation in ice cold HBSS, dissociated with trypsin 0.05% for 30 min to obtain a single cells suspension and reseeded in 100% fresh Matrigel. Organoid cultures were maintained for at least 9 months passaging every 10 days. For analysis, organoids were recovered from Matrigel as before and processed for immunofluorescence or for protein or RNA extraction.
For the experiment depicted in
Immunofluorescences on PFA-fixed samples were performed as previously described (ordenonsi, M. et al. The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 147, 759-772 (2011)). Briefly, samples were fixed 10 min at room temperature with 4% PFA solution. Slides were permeabilized 10 min at RT with PBS 0.3% Triton X-100, and processed for immunofluorescence according to the following conditions: blocking in 10% Goat Serum (GS) in PBS 0.1% Triton X-100 (PBST) for 1 hr followed by incubation with primary antibodies (diluted in 2% GS in PBST) overnight at 4° C., four washes in PBST and incubation with secondary antibodies (1:200 in 2% GS in PBST) for 2 hours at room temperature. Samples were counterstained with ProLong-DAPI (Molecular Probes, Life Technologies) to label cell nuclei.
For immunofluorescence on mammary organoids (
For immunofluorescence on mammary and brain tissue, biopsies were fixed with PFA, paraffin-embedded and cut in 10 μm-thick sections. Sections were re-hydrated and antigen retrieval was performed by incubation in citrate buffer 0.01 M pH 6 at 95° C. for 20 minutes. Slides were then permeabilized (10 min at RT with PBS 0.3% Triton X-100 for mammary sections and 10 min at RT with PBS 1% Triton X-100 for brain sections) and processed as described above.
Primary antibodies: anti-YAP (4912; 1:25) polyclonal antibody, anti-CNPase (5664S; 1:100) polyclonal antibody, anti-SOX2 (4900; 1:50) monoclonal antibody were from Cell Signaling Technology. anti-TAZ (anti-WWTR1, HPA007415; 1:25) polyclonal antibody, anti-α-SMA (A2547; 1:400) mouse monoclonal antibody and anti-amylase (A8273:1:200) rabbit polyclonal antibody were from Sigma. anti-TuJ1 (anti β-III-tubulin; MMS435P-100; 1:500) mouse monoclonal antibody was from Covance. anti-GFAP (Z0334; 1:1000) rabbit polyclonal antibody was from Dako. anti-Nestin (MAB353; 1:300) mouse monoclonal antibody and anti-Sox9 (AB5535; 1:200) rabbit polyclonal antibody were from Millipore. anti-E-cadherin (610181; 1:1000) monoclonal antibody was from BD Biosciences. anti-K14 (Ab7800; 1:100) mouse monoclonal antibody, anti-NeuN (Ab177487; 1:100) rabbit monoclonal antibody, anti-K8 (Ab14053; 1:100) chicken polyclonal antibody and anti-GFP (Ab13970; 1:100) polyclonal antibody were from Abcam. anti-GFP (A6455; 1:100) rabbit serum was from Life Technologies. anti-p63 (H137, sc-8343; 1:50) and anti-Vimentin (Vim C-20, sc-7557-R; 1:100) rabbit polyclonal antibodies were from Santa Cruz. anti-Tau (1:100) rabbit polyclonal antibody was from Axell. K19 was detected using the monoclonal rat anti-Troma-III antibody (DSHB; 1:50). Alexa-conjugated secondary antibodies (Life Technologies): Alexa-Fluor-488 donkey anti-mouse IgG (A21202); Alexa Fluor-568 goat anti-mouse IgG (A11031); Alexa-Fluor-647 donkey anti-mouse (A31571); Alexa Fluor-488 goat anti-mouse IgG2a (A21131), Alexa Fluor-647 goat anti-mouse IgG1 (A21240), Alexa Fluor-488 donkey anti-rabbit IgG (A21206), Alexa-Fluor-568 goat anti-rabbit IgG (A11036), Alexa-Fluor-647 donkey anti-rabbit IgG (A31573); Alexa Fluor-555 goat anti-chicken IgG (A21437). Goat anti-rat Cy3 (112-165-167) was from Jackson Immunoresearch.
For X-gal staining (
Confocal images were obtained with a Leica TCS SP5 equipped with a CCD camera. Bright field and native-GFP images were obtained with a Leica DM IRB inverted microscope equipped with a CCD camera (Leica DFC 450C). Live cell imaging was performed with a A1Rsi+laser scanning confocal microscope (Nikon) equipped with NIS-Elements Advanced Research Software.
Western blots were carried out according to standard procedures. Anti-YAP/TAZ (63.7; sc-101199) and anti-p63 (4A4; sc-8431) monoclonal antibodies were from Santa Cruz. anti-GAPDH (MAB347) monoclonal antibody was from Millipore. Anti-K14 (Ab7800) mouse monoclonal antibody and anti-K8 (Ab14053) chicken polyclonal antibody were from Abcam.
Quantitative Real-Time PCR (qRT-PCR)
Cells or tissues were harvested in TriPure (Roche) for total RNA extraction, and contaminant DNA was removed by DNase treatment. qRT-PCR analyses were carried out on retrotranscribed cDNAs with Rotor-Gene Q (Quiagen) thermal cycler and analyzed with Rotor-Gene Analysis6.1 software. Expression levels are always given relative to Gapdh, except for
For microarray experiments, Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, Calif., USA) were used. Total RNA was extracted using TriPure (Roche) from:
1) luminal differentiated mammary cells (3 replicas), organoids derived from yMaSCs (3 replicas), and MaSCs (3 replicas);
2) cortical neurons (3 replicas), yNSCs (from YAP wild type-transduced cortical neurons, passage 2; 3 replicas), and native NSCs (3 replicas);
3) pancreatic exocrine acini (4 replicas), yDucts (passage 10; 4 replicas), and Ducts (passage 10; 4 replicas).
RNA quality and purity were assessed on the Agilent Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany); RNA concentration was determined using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc.). RNA was then treated with DNaseI (Ambion). In vitro transcription, hybridization and biotin labeling were performed according to Affymetrix 3′IVT protocol (Affymetrix). As control of effective gene modulation and of the whole procedure, we monitored the expression levels of tissue-specific markers of differentiated cells or stem/progenitors by qRT-PCR prior to microarray hybridization and in the final microarray data.
All data analyses were performed in R (version 3.1.2) using Bioconductor libraries (BioC 3.0) and R statistical packages. Probe level signals were converted to log 2 expression values using robust multi-array average procedure RMA46 of Bioconductor affy package. Raw data are available at Gene Expression Omnibus under accession number GSE70174. Global unsupervised clustering was performed using the function hclust of R stats package with Pearson correlation as distance metric and average agglomeration method. Gene expression heatmaps have been generated using the function heatmap.2 of R gplots package after row-wise standardization of the expression values. Before unsupervised clustering, to reduce the effect of noise from non-varying genes, we removed those probe sets with a coefficient of variation smaller than the 90th percentile of the coefficients of variation in the entire dataset. The filter retained 4511 probe sets that are more variable across samples in any of the 3 subsets (i.e., mammary, neuron, and pancreatic).
C57BL/6J mice and NOD-SCID mice were purchased from Charles River. Transgenic lines used in the experiments were gently provided by: Duojia Pan (Zhang, N. et al. The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev Cell 19, 27-38 (2010)) (Yap1fl/fl and R26-LSL-LacZ); Cedric Blanpain (K8-CreERT2/R26-LSL-YFP) (Van Keymeulen, A. et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189-193 (2011)); Doron Merckler (Thy1-Cre)(Dewachter, I. et al. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. The Journal of neuroscience: the official journal of the Society for Neuroscience 22, 3445-3453 (2002)); Ivan De Curtis (Syn1-Cre)(Zhu, Y. et al. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes & development 15, 859-876 (2001)); Giorgio Carmignoto (R26-CAG-LSL-tdTomato) (Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nature neuroscience 13, 133-140 (2010)); Fernando Camargo (tetO-YAPS127A) (Camargo, F. D. et al. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr Biol 17, 2054-2060 (2007)). Tazfl/fl and double Yapfl/fl; Tazfl/fl conditional knock-out mice were as described in (Azzolin, L. et al. YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response. Cell 158, 157-170 (2014)). Ptf1a-CreERTM (stock #019378), R26-LSL-rtTA-IRES-EGFP (stock #005670) and R26-rtTAM2 mice (stock #006965) were purchased from The Jackson Laboratory. Animals were genotyped with standard procedures and with the recommended set of primers. Animal experiments were performed adhering to our institutional guidelines as approved by CEASA.
To obtain Thy1-Cre; R26-LSL-LacZ/+ mice, we crossed Thy1-Cre hemizygous males with R26-LSL-LacZ/LSL-LacZ females. Littermate embryos derived from these crossings were harvested at E18-19 and kept separate for neurons/NSCs derivation; genotypes were confirmed on embryonic tail biopsies.
To obtain Thy1-Cre; R26-LSL-rtTA-IRES-EGFP/+ mice, we crossed Thy1-Cre hemizygous males with R26-LSL-rtTA-IRES-EGFP/LSL-rtTA-IRES-EGFP females. Littermate embryos derived from these crossings were harvested at E18-19 and kept separate for neurons derivation; genotypes were confirmed on embryonic tail biopsies.
To obtain Syn1-Cre lineage tracing studies, we used Syn1-Cre hemizygous females (as transgene expression in male mice results in germline recombination (Rempe, D. et al. Synapsin I Cre transgene expression in male mice produces germline recombination in progeny. Genesis 44, 44-49 (2006))) with R26-LSL-rtTA-IRES-EGFP homozygous males or R26-CAG-LSL-tdTomato/+ males. Littermate embryos derived from these crossings were harvested at E18-19 and kept separate for neurons derivation; genotypes were confirmed on embryonic tail biopsies.
To obtain R26-rtTAM2; tetO-YAPS127A mice, we crossed R26-rtTAM2/+ mice with tetO-YAPS127A mice. R26-rtTAM2/+ littermates were used as negative control.
To obtain Ptf1a-CreERTM; R26-LSL-rtTA-IRES-EGFP/+; tetO-YAPS127A mice, we crossed Ptf1a-CreERTM; R26-LSL-rtTA-IRES-EGFP/LSL-rtTA-IRES-EGFP mice with tetO-YAPS127A mice. Ptf1a-CreERTM; R26-LSL-rtTA-IRES-EGFP/+ littermates were used as negative control.
The number of biological and technical replicates and the number of animals are indicated in Fig. legends and specification. All tested animals were included. Animal ages are specified in the specification. Sample size was not predetermined. Experiments were performed without methods of randomization or blinding. For all experiments with error bars the standard deviation (s.d.) was calculated to indicate the variation within each experiment.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2015/070305 | 9/4/2015 | WO | 00 |
