t cell development is unique among all hematopoietic lineages ; it  requires a distinct organ , the thymus .
thymic epithelial cells ( tecs ) provide unique structural and functional niches , which allow for t  cell lineage induction , somatic generation , and subsequent selection (  quality control  ) of the nascent t  cell repertoire ( anderson and takahama , 2012 ) .
the two major subsets of tecs  cortical ( ctecs ) and medullary tecs ( mtecs)define the two structural compartments of the thymus , the cortex and the medulla .
t  cells migrate during their development through both compartments in a spatially and temporally ordered process . during the cortical phase ,
a highly diverse t  cell repertoire is generated in a random fashion and subjected to positive selection for self - mhc restriction .
the subsequent medullary phase imposes t  cell tolerance on the nascent repertoire via negative selection of autoreactive effector cells and differentiation of regulatory t  cells ( heng et  al . , 2010 ;
failure of developing and/or maintaining an intact and functional thymic epithelial compartment can result either in complete t  cell deficiency as exemplified by mutations of the transcription factor foxn1 or in a skewed t  cell repertoire predisposing to autoimmunity as observed in various mutants affecting the nf-b pathway ( tykocinski et  al .
thymus development starts around embryonic day 10.5 ( e10.5 ) , when parts of the ectodermal cervical vesicle come into close contact with the pharyngeal endoderm .
the budding and the outgrowth of the thymic anlage occur at e11.5 , which is also the onset of foxn1 expression in these endodermal cells .
the first hematopoietic colonization occurs around e11.5 and the delineation of the cortex and medulla compartments becomes apparent at e14 ( gordon and manley , 2011 ) .
the thymus then increases in size until weaning and after puberty slowly and steadily involutes . in the postnatal thymus
for instance , mature mtecs have a half - life of approximately 2  weeks ( gbler et  al . , 2007 ;
these observations suggest the existence of self - renewing stem and/or progenitor cells replenishing the mature mtec subset . indeed , clonogenic , medullary islet - forming mtec progenitors have been identified ( rodewald et  al . , 2001 ;
in addition , proliferating ctec progenitors have been characterized in the fetal thymus ( shakib et  al . , 2009 ) .
it is presumed that both lineage - committed precursor pools arise from a bipotent tec progenitor / stem cell ( bleul et  al . , 2006 ; rossi et  al . ,
attempts to identify , characterize , and prospectively purify these bipotent tec progenitor / stem cells have so far met with limited success , and the phenotype of tec stem cells still remains to be defined ( boehm , 2008 ; baik et  al . , 2013 ) . yet , the existence of embryonic bipotent tec progenitors , which could give rise to both cortical and medullary progeny , has been demonstrated in a single - cell transplantation assay ( rossi et  al . , 2006 ) .
in  vivo cell lineage tracing revealed the persistence of dormant embryonic tec progenitors in the postnatal thymus , which still could initiate the formation of a functionally competent minithymus ( bleul et  al . , 2006 ) .
colony - forming , multipotent thymic cells have also been isolated from the postnatal rat thymus ( bonfanti et  al . , 2010 ) .
however , whether these bipotent progenitors bear the stemness features including self - renewal and low cycling rate remained unclear .
moreover , without the ability to prospectively enrich for thymic epithelial stem cells , the assessment of their developmental potential in  vitro or in  vivo at the single - cell level has not been possible .
one experimental approach to characterize epithelial stem cell populations ex  vivo exploits their ability to form spheroid colonies in the presence of specific growth factors under low - attachment culturing conditions .
this method had been first established for neuronal stem cells ( neurospheres ) ( reynolds and weiss , 1996 ) and later been adapted to other tissues of epithelial origin ( e.g. , of the mammary gland ) ( dontu et  al . , 2003 ) . under the culture conditions of sphere formation ,
thus , the sphere - culture  method provides a valuable single - cell assay to isolate , characterize , and quantify stem cells with sphere - forming capacity in a species - independent manner ( pastrana et  al . , 2011 ) . here
we report that thymic stromal cells of the adult mouse thymus contain rare sphere - forming cells and document that these cells bear the stemness features of low cycling rate , self - renewal , and bipotency , i.e. , giving rise to both the ctec and mtec lineages , thereby characterizing them as thymic epithelial stem cells .
moreover , we show that the transcription factor foxn1 is dispensable for the maintenance and the lineage commitment of these thymic epithelial stem cells .
in analogy to sphere - forming stem cells identified in various other epithelial tissues , we sought to determine whether nonhematopoietic thymic stromal cells also contain a similar cell type . to this end
we isolated the thymic stromal cell - enriched fraction from the dissected thymi of adult mice via sequential enzymatic dissociation and subsequent depletion of cd45 cells and cultured them under low attachment conditions in the presence of  epidermal growth factor ( egf ) and basic fibroblast growth factor  ( bfgf ) ( see experimental procedures ) .
indeed , spherical structures reminiscent of neurospheres or mammospheres were formed within 7  days in  vitro ( figures 1a and 1b ) .
thymospheres .  of note , the appearance of thymospheres was a slow and progressive process ( figure  s1a ) , suggesting that their formation was not simply due to the aggregation of the cultured cells .
instead , thymosphere formation apparently requires several rounds of cell divisions as observed in other sphere - forming systems . to verify that thymospheres were derived from a single sphere - forming cell through several rounds of cell division , we performed a label retention assay using the pkh-26-labeling method as described recently ( pece et  al . , 2010 ) .
pkh-26 is a lipophilic membrane dye that integrates into the cytoplasmic membrane of all cell types . during subsequent culturing of the labeled cells in the absence of the dye
, the amount of incorporated pkh-26 on cellular membranes will be halved with each cell division .
thus , only slow - cycling cells will retain the pkh-26 fluorescence . using this assay
it has been shown earlier for mammospheres that at the onset of sphere formation , the sphere - forming cell divides only once in an asymmetric fashion , giving rise to a mitotic bipotent progenitor cell and a quiescent sphere - forming cell ( pece et  al . , 2010 ) .
other cells subsequently generated during the sphere formation arise from this mitotic progenitor cell and thereby serially dilute the pkh-26 label , whereas only the rarely dividing stem cell retains the pkh-26 fluorescence .
similarly , using pkh-26-labeled primary thymic stromal cells , we observed that the thymospheres consisted of a single pkh-26 cell surrounded by a number of pkh-26 cells ( figure  1c ) .
this result demonstrates that thymospheres formed in these cultures originated from a label - retaining and slow - cycling cell , referred to here as the thymosphere - forming cell ( tsfc ) .
furthermore , this result also rules out the possibility of aggregation as the cause of the observed sphere formation , because aggregation of pkh-26-labeled cultured cells should generate structures containing cells that are equally pkh-26 , which has never been observed in our thymosphere cultures .
additionally , we performed a mixing experiment where primary thymic cells obtained from two transgenic mouse lines ( rosa - tmtomato and cag - gfp - tg ) were mixed in equal parts and cultured for sphere formation .
the presence of gfp - only and tomato - only thymospheres in these cultures provides additional support for the clonality of the formed thymospheres ( figure  s1b ) .
to determine the frequency of the tsfcs within the isolated primary adult thymic stromal cells , we performed extreme limiting dilution analyses using the elda software ( figure  1d ; hu and smyth , 2009 ) .
the tsfc frequency was predicted to be 1 in 1,493 nonhematopoietic cells ( nhcs ) within the 95% confidence interval of 1/2,405 to 1/926 nhcs .
the lack of fit test for single - hit model gave an insignificant p value of 0.196 , indicating that a thymosphere is formed by a single cell , i.e. , the tsfc . this result
also clearly rules out aggregation of the plated cells as an explanation of the observed spheres because this would follow a multihit model . taken together
, our results demonstrated that the adult thymus contains rare sphere - forming cells similar to the ones isolated from various other organs including the mammary gland and the forebrain .
several stemness features distinguish tissue - restricted stem cells from transit - amplifying progenitors and terminally differentiated cells of the corresponding tissue : ( 1 ) low cycling rate , ( 2 ) self - renewal capacity , and ( 3 ) multipotentiality , i.e. , the ability to give rise to distinct tissue - specific cell lineages .
accordingly , we asked whether tsfcs bear stemness features and thereby represent thymic epithelial stem cells .
first , tsfcs have low mitotic activity as demonstrated in  vitro by the pkh-26 label retention assay ( figure  1c ) .
second , we assessed the self - renewal capacity of the thymosphere - forming cells in  vitro by their ability to re - form spheres upon replating of dissociated primary spheres .
thymospheres generated from 5-week - old mouse thymi were size - selected on 35  m filters ( thereby the residual single cells were removed ) , dissociated into single cells , and recultured , which resulted in the formation of secondary spheres ( figure  2a ) .
tertiary and quaternary spheres were generated in the same manner in subsequent steps of cultivation , dissociation , and sphere re - formation ( figure  2a ) .
the sphere formation assay has been applied to various epithelial cell lineages as a valuable approach to enrich for stem cells .
indeed , quantification of the tsfc frequency in thymosphere re - formation assay demonstrated a 8.8-fold enrichment of tsfcs upon primary sphere formation ( figure  2b ) .
the tsfc frequency then slightly decreased in the third passage compared to secondary sphere formation as had also been reported for mammospheres ( pece et  al . , 2010 ) . when thymic stromal cells were only initially labeled with pkh-26
after the dissociation of the thymus and the sphere re - formation assay was repeated ( figure  s2 ) , we observed a single pkh-26 cell in both secondary and tertiary thymospheres ( figures 2c and 2d ) .
this result shows that re - formed spheres were derived from the pkh-26 label - retaining cells of the previous passage spheres , thus documenting that thymosphere - forming cells indeed have self - renewal capacity .
next we assessed tsfcs for bipotentiality , i.e. , their ability to give rise to both the mtec and ctec lineages .
commitment into  either mtec or ctec lineages was assessed by whole - mount immunostaining of primary thymospheres for cytokeratins 8 and 14 ( k8 and k14 ) .
confocal microscopy images demonstrated that thymospheres contained cells expressing either an mtec lineage marker profile ( k14k8 ) or a ctec lineage profile ( k14k8 ) ( figures 3a and s3 ) .
the presence of lineage - committed mtecs  and ctecs within thymospheres clearly demonstrates the bipotentiality of the tsfcs .
we therefore analyzed the expression of previously described markers of bipotent tec progenitors : cd205 ( also expressed by terminally differentiated ctecs ) ( baik et  al .
, 2013 ) , 5 t ( also expressed by terminally differentiated ctecs ) ( ohigashi et  al . , 2013 ) , and cytokeratin 5 ( k5 ; also expressed by terminally differentiated mtecs ) ( klug et  al . , 1998 ) .
whole - mount coimmunostaining of primary thymospheres for k14 and cd205 ( figure  3b ) showed that almost all k14 cells expressed cd205 .
when we performed k14 immunostaining using primary thymospheres generated from thymic stromal cells isolated from a transgenic mouse line expressing venus ( gfp ) under the control of 5 t promoter , most of the k14 cells showed 5 t promoter activity ( figure  3c ) .
we also generated thymospheres using primary thymic stromal cells isolated from a transgenic mouse line expressing the h2b - gfp fusion protein under the control of k5 promoter .
confocal microscopy analyses of these thymospheres after immunostaining for k8 demonstrated that there were high numbers of k5k8 cells in these thymospheres ( figure  3d ) .
in addition to these markers , we assessed the expression of epithelial ( pan - ck ) ( figure  3e ) , endothelial ( cd31 ) ( figure  3f ) , and mesenchymal ( vimentin ) ( figure  3 g ) cell markers within the thymospheres .
expectedly , all cells within the thymospheres were cytokeratin positive , and no cd31- or vimentin - expressing cells were detected in any of the analyzed thymospheres .
these results demonstrate that the thymospheres are generated by bipotent thymic epithelial stem cells and are composed of both  lineage - committed and -uncommitted thymic epithelial cells .
collectively , our results demonstrate that tsfcs bear all three genuine stemness features and therefore represent adult thymic epithelial stem cells .
to analyze whether the thymic epithelial progenitor cells within  thymospheres can generate tecs with their typical morphologies in monolayer cultures , we dissociated the isolated primary thymospheres into single - cell suspensions and cultured them on collagen - coated coverslips .
after 9  days of culture , large and flat cells with thymic epithelial morphology were observed , which were either k14 ( mtecs ) or k8 ( ctecs ) ( figure  4a ) .
next , we assessed the potential of thymospheres to form ctecs and mtecs in an in  vivo assay . to this end
thymospheres derived from genetically marked animals , harboring ubiquitous expression of the tomato fluorescent protein , were mixed with wild - type cells derived from e14 embryonic thymi in reaggregation thymic organ cultures ( rtocs ) .
after 1  day in  vitro culture , reaggregates were transplanted under the kidney capsule of recipient nude mice . at 46  weeks after transplantation ,
a subset of these epcam donor cells upregulated the maturation marker mhcii , indicative of development into mature tecs ( figure  4b ) .
tomato cells seeded both the cortex and the medulla of the transplant , where they coexpressed cortical ( k8 ) and medullary ( k5 ) lineage markers , respectively ( figures 4c and 4d ) . because uea1 or aire cells represent the terminally differentiated functional mtecs ( gbler et  al .
we were able to detect tomatouea1 ( figure  4e ) and tomatoaire ( figure  4f ) cells in the medullary region of the transplant , indicating that sphere - derived tecs were functional and underwent proper terminal differentiation .
taken together , these results demonstrate that the tsfcs and tec progenitors within the thymospheres can give rise to terminally differentiated medullary and cortical tecs both in  vitro and upon long - term reconstitution in  vivo .
the low abundance of tsfcs within isolated primary thymic cells  prompted us to identify surface markers allowing for their prospective enrichment .
surprisingly , cd45epcam cells did not form thymospheres , whereas the cd45epcam cell population contained all tsfcs ( figure  5a ) . because not all epithelial cells express epcam , but epithelial cell identity is rather defined by cytokeratins ( moll et  al .
, 2008 ) , we assessed cytokeratin expression in primary thymic cells by flow cytometry using a pan - cytokeratin antibody .
indeed , the cd45epcam cells contained a distinct cytokeratin - positive population ( figure  5b ) , demonstrating that thymic epithelial cells also reside in the epcam fraction of thymic stromal cells .
we next subdivided the cd45epcam population according to the expression of sca1 ( stem cell antigen 1 ) and cd24 ( also known as heat - stable antigen ) , both of which have been described as stem cell markers in other tissues ( welm et  al .
, 2002 ; shackleton et  al . , 2006 ; stingl et  al . ,
2006 ) . only the sca1cd24 population formed thymospheres , whereas the other populations ( sca1cd24 , sca1cd24 , and sca1cd24 ) were devoid of tsfcs ( figure  5c ) .
based on these results we obtained a 4.6-fold enrichment of tsfcs compared to the total thymic stromal cell fraction ( cd45 population ) via sorting the cd45epcamsca1cd24 subset ( figure  5d ) .
next we performed an unbiased surface marker screen ( bd ,  lyoplate assay ) evaluating the expression profile of 176 different surface antigens on pregated cd45epcamsca1cd24 stromal cells ( figure  s4a ) .
antigens displaying a relative distribution of 1:3 or more were considered potentially useful for further tsfc enrichment . based on the outcome of this screen
, the cd45epcamsca1cd24 population was subdivided according to the expression of the surface markers cd38 or cd31 .
in addition we analyzed the surface marker cd49f , a known stem cell marker in other systems , e.g. , mammary gland ( stingl et  al . , 2006 ) .
for all three antigens the positive and negative fractions  were assessed for thymosphere formation .
for all these markers , the tsfcs clearly partitioned into the respective negative fractions ( figures s4b  s4d ) .
based on these results , we designed a lineage cocktail ( lin ) including biotin - labeled antibodies directed against cd45 , epcam , cd31 , cd38 , and cd24 .
sorting for linsca1 cells resulted in a 6.7-fold enrichment of thymosphere - forming cells over total cd45 thymic stromal cells ( figures 5e and 5f ) .
this amount of enrichment corresponds to a  ratio of 1 tsfc in 296 linsca1 cells and  thereby allowed us to perform limiting - dilution experiments resulting in sphere formation even when only 10 linsca1 cells were plated per well ( data not shown ) .
taken together , our results demonstrate that tsfcs express a distinct surface antigen profile within the thymic stroma and can be highly enriched as a linsca1 cell population .
expression of the foxn1 transcription factor in tecs is indispensable for thymus development , because its activity is required for the generation and maintenance of functional mature mtecs and ctecs ( gordon and manley , 2011 ) . in the absence of a functional foxn1 ,
e.g. , in nude mice or foxn1-deficient mice , thymic development is arrested , which leads to the abolishment of t  cell development and thus an immunodeficiency phenotype ( rodewald 2008 ) .
earlier studies suggested that foxn1 expression in the thymic primordium at e11.25 specifies thymic epithelial identity ( gordon et  al .
, 2001 ) . however , reconstitution of foxn1 expression in postnatal foxn1-deficient mice resulted in the development of a functional minithymus , indicating that tec progenitors were still present in the thymic rudiment of foxn1-deficient mice even though they were not able to develop into ctecs and mtecs ( bleul et  al . , 2006 ) .
recent studies provide indirect evidence suggesting that foxn1 might be dispensable for commitment into the mtec and ctec lineages but nevertheless is required for their subsequent differentiation ( nowell et  al . , 2011 ) .
based on these findings , we asked whether the thymic rudiment of nude mice would harbor tsfcs .
indeed , the nude thymic rudiment contained tsfcs ( figure  6a ) , clearly demonstrating that the maintenance of thymic epithelial stem cells does not require the function of foxn1 .
next , we performed lineage - tracing experiments in order to determine whether thymosphere - forming cells reside in the foxn1 or foxn1 lineage . to this end
we generated a double transgenic mouse line , in which foxn1 and foxn1 lineages were marked by the expression of tomato and gfp , respectively , whereby gfp expression was indelibly induced by cre - recombinase expression under the control of the endogenous foxn1 promoter ( figure  6b ) .
the tsfcs were found exclusively in the tomato fraction , i.e. , in the foxn1 lineage , and the foxn1 lineage ( gfp ) did not form any thymospheres ( figure  6c ) .
foxn1 expression was not induced even during sphere formation ; all cells within these spheres remained tomato and gfp ( figure  6d ) .
as shown above , thymospheres contained lineage - committed cells of both the mtec and ctec lineages ( figures 3a and s3 ) .
the lack of foxn1 upregulation in the sphere culture therefore implies that foxn1 expression may not be required for mtec and ctec  lineage commitment .
hence , we analyzed thymospheres derived from the nude thymic rudiments by immunostaining for mtec and ctec lineage markers and demonstrated that k14k8 mtec and k8k14 ctec lineages emerged in these spheres despite the absence of a functional foxn1 transcription factor ( figures 6e and s5 ) . nevertheless , because foxn1 is important for the functional development and maintenance of committed tecs , we hypothesized that upregulation of foxn1 might take place during tec differentiation after lineage commitment had occurred .
indeed , when spheres derived from foxn1 lineage were dissociated and plated under in  vitro differentiation culture conditions , we observed a switch from tomato to gfp expression , indicative of foxn1 promoter activity ( figure  6f ) . taken together , these results document that foxn1 is dispensable both for the maintenance of the adult thymic epithelial stem cell pool in  situ and for the molecular events underlying mtec and ctec lineage commitment processes . therefore ,
thymic epithelial cells furnish essential niches for t  cell development and for the establishment of central immune tolerance .
the cellular and molecular mechanisms underlying early steps in tec ontogeny and subsequent commitment into both the ctec and mtec lineages are still poorly understood .
an important missing link in studies on the organogenesis and regeneration of the thymus has been the unambiguous identification and characterization of thymic epithelial stem cells .
several previous studies reported on the identification of early bipotent thymic epithelial progenitors within the thymus of fetal or newborn rodents using different experimental approaches ( bleul et  al . , 2006 ;
rossi et  al . , 2006 ; bonfanti et  al . , 2010 ) . because these studies focused on bipotentiality but did not further characterize the progenitor cells , which potentially gave rise to both lineages , it remained unclear whether those bipotent cells displayed bona fide stemness features .
here we isolated thymic epithelial stem cells from the adult thymus on the basis of their sphere - forming potential in ultralow attachment conditions , a conserved feature of stem cells of various epithelial tissues .
we demonstrate that the thymospheres are formed by a single label - retaining cell , indicating that thymosphere - forming cells are slow cycling , a common feature of tissue - restricted stem cells .
indeed , tsfcs self - renew as shown in  vitro by several consecutive rounds of thymosphere dissociation and subsequent sphere reformation .
finally , we document that thymospheres contain cells expressing either an mtec ( k14k8 ) or ctec ( k8k14 ) lineage marker profile .
because all cell types within a thymosphere are descendants of the tsfc , this result indicates that the tsfcs are bipotent .
furthermore , we performed whole - mount immunostaining of thymospheres for various putative markers of bipotent tec progenitors reported in earlier studies , such as 5 t , cd205 , and k5 .
the results demonstrate that thymospheres contain both lineage - committed tecs ( k8 or k14 single - positive cells ) and uncommitted bipotent tec progenitors .
additional experiments using in  vitro differentiation cultures and transplantation of rtocs demonstrate that cells derived from thymospheres can further differentiate and generate the corresponding mtecs and ctecs both in  vitro and in  vivo . the expression of mhcii , aire , or uea1 by the transplanted thymosphere - derived cells indicate that the tecs generated from thymospheres are functionally competent in  vivo .
hence , tsfcs of adult murine thymi bear three canonical hallmarks of stemness and thereby qualify as thymic epithelial stem cells . using thymosphere formation as a readout , we determined the surface antigen profile of these thymic epithelial stem cells as linsca1 , whereby lin refers to a lineage cocktail of antigens including cd45 , ter119 , epcam , cd24 , cd31 , and cd38 .
importantly , during the identification of the surface antigen profile of tsfcs , we applied stringent selection criteria in order to determine those subpopulations that exclusively contained tsfcs .
based on this surface antigen profile , these rare tec stem cells can now be highly enriched from dissociated thymic stromal cells of adult mice . of note , we can not rule out the possible existence of another epithelial stem cell pool within the adult thymus that does not form thymospheres under our culturing conditions . in such cases ,
the determined surface antigen profile presented in this study may refer only to the sphere - forming thymic epithelial stem cells .
the finding that tsfcs reside exclusively in the epcam fraction  of the thymic stroma was somewhat surprising because epcam expression is commonly considered to be the distinguishing marker of epithelial cells within the thymus .
however , epcam epithelial cells have been described in other tissues ( e.g. , differentiated keratinocytes ) ( trzpis et  al . , 2007 ) .
moreover , epcam expression has been also detected in nonepithelial cells , such as thymocytes and es cells , not yet committed to  epithelial lineage ( trzpis et  al . , 2007 ) , which suggests that epcam expression can not be considered as a bona fide marker of epithelial cell identity .
in contrast , cytokeratin expression unambiguously marks epithelial identity because all epithelial cells express a pair of acidic and basic cytokeratins ( moll et  al . , 2008 ) .
in this respect , cytokeratin expression by a subset of epcam cells suggests that there are indeed epcam epithelial cells within the thymic stroma .
this finding calls for a re - evaluation of epcam as an appropriate tec marker , at least as far as stem / progenitor cells are concerned . of note ,
epithelial stem cells of the mammary gland were also shown to be epcam ( eirew et  al . , 2008 ; lim et  al . ,
foxn1 is a transcription factor that is indispensable for the generation of a functional thymic epithelial compartment .
indeed , the loss of function of foxn1 , as first observed in nude mice , results in thymic aplasia and consequent t  cell deficiency ( nehls et  al . , 1994 ; rodewald , 2008 ) .
recent studies provided indirect evidence for foxn1 being dispensable for the maintenance of embryonic thymic epithelial stem cells and lineage commitment into either mtecs or ctecs ( bleul et  al .
instead , foxn1 is apparently required for transition of already committed tecs from early to late differentiation stages and the maintenance of the postnatal tec compartment ( nowell et  al .
, 2011 ; chen et  al . , 2009 ; cheng et  al . , 2010 ) .
we readdressed the function of foxn1 in the context of sphere - forming adult thymic epithelial stem cells .
it was previously suggested that the thymic rudiment of nude mice still contains putative thymic epithelial stem cells , because stochastic activation of a functional foxn1 gene postnatally led to the development of a fully competent minithymi , thus reverting at least in part thymic atrophy ( bleul et  al . , 2006 ) . as a direct proof of this claim ,
we show that the thymic rudiment of nude mice does contain sphere - forming thymic epithelial stem cells .
interestingly , as analyzed by lineage tracing , adult thymosphere - forming cells are exclusively present within the foxn1 lineage , thus indicating that foxn1 is not yet expressed in stem cells but only at later stages .
this finding concurs with the presence of thymic epithelial stem cells within the thymic rudiment of nude mice , because the loss of function of foxn1 would not directly affect the stem cell compartment .
importantly , although foxn1 was not upregulated in any cell of thymospheres , commitment into mtec and ctec lineages did take place within spheres .
furthermore , mtec and ctec lineage commitment similarly took place in foxn1 mutant ( nude ) thymospheres .
we observed foxn1 promoter activity only during further differentiation in in  vitro differentiation culture of dissociated thymospheres , in line with a role of foxn1 during later stages of tec differentiation .
taken together , our results document that foxn1 function is dispensable both for the maintenance of adult thymic epithelial stem cells and their lineage commitment into mtec and ctec progenitors .
although in this study we focused on the functional characterization of tsfcs within the young adult thymus , similar sphere - forming cells exist in the embryonic thymus as well as in the involuted thymus of 18-month - old mice .
the comparative quantification of sphere frequency ( figure  s1c ) demonstrated that the ratio of tsfcs is significantly higher in the embryonic than in postnatal thymus .
the relative frequency of tsfcs did not significantly change between young adult and involuted thymus . in this respect
, it will be of considerable interest to compare the phenotype and function of tsfcs isolated from a wide range of different age groups in order to address open issues related to embryonic development of the thymus versus adult tec maintenance and mechanisms underlying age - related thymic involution .
similar questions can also be addressed in humans , because thymosphere - forming cells also exist in the postnatal human thymus ( figure  s1d ) .
this will be of particular interest from a translational point of view , because cellular replacement therapies for the treatment of immunodeficiency or autoimmune diseases associated with a dysfunctional thymus would be highly desirable . in this respect , the clonal thymosphere method might present an attractive avenue to enrich for human thymic epithelial stem cells .
when not indicated otherwise , 4- to 6-week - old wild - type c57bl6/n mice were used for the isolation of thymic stromal cells . for rtoc experiments , e14.5 embryos were obtained from time - mated c57bl6/n females .
nmri nude / nude 6-week - old females were used for the isolation of thymic stromal cells and as hosts in rtoc transplantation experiments .
foxn1 knockin mice were kindly provided by n. manley ( university of georgia ) ( foxn1tm3(cre)nrm ) ( liston et  al . , 2007 ) .
rosa - tomato - gfp animals ( b6.129(cg)-gt(rosa)26sortm4(actb - tdtomato ,- egfp)luo / j ) ( muzumdar et  al . , 2007 ) were kindly provided by r. sandhoff ( dkfz ) .
cag - gfp - tg animals were kindly provided by e. erikci and k. chowdhury ( mpi for biophysical chemistry ) .
5t - venus animals ( b6-psmb11tm1smta ) ( murata et  al . , 2007 ) were kindly provided by s. murata ( university of tokyo ) .
k5-h2b - gfp animals ( tg(krt5-tta)#glk tg(teto - hist1h2bj / gfp)47efu / j ) ( tumbar et  al . , 2004 ) were kindly provided by p. angel ( dkfz ) and t. barthlott ( university of basel ) .
all animal experiments were approved by the regional authorities ( regierungsprsidium karlsruhe , germany ) and performed according to the guidelines of the german cancer research center ( dkfz ) .
human thymi were obtained from male and female children under 3 years of age in the course of corrective cardiac surgery at the department of cardiac surgery , medical school of the university of heidelberg , germany .
studies on human samples were approved by the institutional review board of the university of heidelberg .
thymic stromal cell isolation from adult animals and human donors were performed as previously described ( ucar et  al . , 2013 ) with the modification of omitting fcs from rpmi medium .
digested thymic tissue was depleted of cd45 cells using cd45 microbeads ( miltenyi biotec ) . for detailed protocol ,
thymic stroma - enriched single - cell suspensions were blocked with anti - fcr mab 2.4g2 and stained for various surface markers ( see supplemental experimental procedures , antibodies ) .
for dead cell exclusion , pi or dapi were used . for intracellular staining ( pan - ck ) ,
cells were fixed after surface marker staining in 1% pfa and permeabilized in 0.02% triton x-100 .
cell sorting and subsequent analysis was performed on a bd facsaria cell sorter and flowjo ( treestar ) , respectively .
live imaging of the thymosphere cultures was performed using a motorized inverted cell observer z1 ( zeiss ) and image analyses for the quantification of thymospheres were done using axiovision software ( zeiss ) . for counting of thymospheres
, we applied a diameter cut - off of minimum 75   65  m for two perpendicular axes . in quantification experiments , we plated each sorted cellular fractions in 96-well plates using at  least  two different 10-fold dilutions ( e.g. , 10 cells / ml and 10 cells / ml ) and in multiple ( up to 8) technical replicates . to avoid false positives due to secondary aggregation during thymosphere formation in the prospectively enriched fractions ( which should contain more tsfcs per well )
, we counted the lowest dilution plated that still contained at least one sphere per replicate ( well ) .
spheres were grown in ultralow attachment plates ( corning ) at variable densities in mebm medium ( lonza ) supplemented with b27 supplement ( gibco ) , 0.5  g / ml hydrocortisone ( sigma ) , 5  g / ml insulin ( sigma ) , 4  g / ml heparin ( sigma ) , 20  ng / ml bfgf ( invitrogen ) , and 20  ng / ml egf ( sigma ) . for precise enumeration of thymospheres under various experimental conditions
pkh-26 labeling was performed on fresh thymic digests using pkh-26 red fluorescent cell linker kit for general cell membrane labeling ( sigma ) at a concentration of 1   10 m pkh-26 dye per 1   10 cells according to the manufacturer s protocol .
imaging of spheres was performed with a motorized inverted cell observer z1 ( zeiss ) . for the sphere re - formation assay ,
thymospheres were collected by size selection on 35  m filters , dissociated into single cells both enzymatically ( 10  min in accumax , sigma ) and mechanically by gentle pipetting , filtered through 35  m filters , and plated in the thymosphere - culturing medium as described above at a density of 10 and 10 cells per ml .
spheres were size - selected on 35  m filter , dissociated enzymatically ( 2  min in  0.05% trypsin , 0.53  mm edta-4na ; invitrogen ) and mechanically by gentle pipetting , and plated onto collagen - coated coverslips in dmem / f12 supplemented with 10% fcs , 5  g / ml insulin ( sigma ) , 0.5  g / ml hydrocortisone ( sigma ) , 20  ng / ml bfgf ( invitrogen ) , 20  ng / ml egf ( sigma ) , and pen / strep .
after 9  days in culture , cells were fixed in 4% pfa in pbs , washed with pbs , permeabilized in 0.5% triton x-100 , and stained for tec lineage markers in pbs / bsa ( 1% bsa in pbs ) .
spheres were fixed in 2% paraformaldehyde , 100  mm hepes , 50  mm egta , 10  mm mgso4 , and 0.05% glutaraldehyde , washed in pbs / bsa , and permeabilized in 0.2%
triton x-100 in pbs / bsa overnight . after blocking in 3% bsa in  pbs ,
spheres were stained at rt with antibodies against various markers ( k8 , k14 , pan - ck , cd205 , vimentin , cd31 ) in pbs / bsa and mounted with prolong gold antifade reagent with dapi ( molecular probes ) .
thymic stroma reaggregates were prepared from fetal thymic stromal cells as previously described ( jenkinson et  al . , 1992 ) .
in brief , thymi derived from e14.5 c57bl/6n embryos were digested with trypsin solution for 5  min at 37c and mixed with thymospheres derived from rosa - tomato - gfp reporter mouse line .
the reaggregates were placed on a 0.8  m nucleopore filter ( whatman ) covering a floating sponge in a medium - filled 6-well plate . for long - term reconstitution assays ,
rtocs were grafted under the kidney capsule of adult nmri nude mice after 1  day of culture ( rodewald et  al . , 2001 ;
the transplants were analyzed 4 and 6  weeks later by flow cytometry or histology .
the stem cell frequency in the limiting dilution experiment was assessed using the elda ( extreme limiting dilution analysis ) software ( http://bioinf.wehi.edu.au/software/elda/ ) ( hu and smyth , 2009 ) .
a 95% confidence interval of active cell frequency was computed for two independent experiments , and single - hit hypothesis was tested by the software using the goodness - of - fit test . for the calculation of statistical significance in enrichment of tsfcs within different sorted cellular fractions , student s t test method was used .
a.u . , o.u . , and p.k . designed and performed the experiments and analyzed and interpreted results .
s.m . performed all confocal analyses and contributed to the sphere re - formation assay . f.b .
 summarywithin the thymus , two major thymic epithelial cell ( tec ) subsets  cortical and medullary tecs  provide  unique structural and functional niches for t  cell development and establishment of central tolerance . 
 both lineages are believed to originate from a common progenitor cell , yet the cellular and molecular identity of these bipotent tec progenitors / stem cells remains ill defined . 
 here we identify rare stromal cells in the murine adult thymus , which under low - attachment conditions formed spheres ( termed  thymospheres  ) . these thymosphere - forming cells ( tsfcs ) displayed the stemness features of  being slow cycling , self - renewing , and bipotent . 
 tsfcs could be significantly enriched based on their distinct surface antigen phenotype . 
 the foxn1 transcription factor was dispensable for tsfcs maintenance in  situ and for commitment to the medullary and cortical tec lineages . in summary 
 , this study presents the characterization of the adult thymic epithelial stem cells and demonstrates the dispensability of foxn1 function for their stemness . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions