Clonal isolation of hESCs reveals heterogeneity within the pluripotent ...

© 2006 Nature Publishing Group http://www.nature.com/naturemethods

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Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment Morag H Stewart1,2, Marc Bosse´2, Kristin Chadwick2, Pablo Menendez1, Sean C Bendall1,2 & Mickie Bhatia1,2 Human embryonic stem cell (hESC) lines are known to be morphologically and phenotypically heterogeneous. The functional nature and relationship of cells residing within hESC cultures, however, has not been evaluated because isolation of single hESCs is limited to drug or manual selection. Here we provide a quantitative method using flow cytometry to isolate and clonally expand hESCs based on undifferentiated markers, alone or in combination with a fluorescent reporter. This method allowed for isolation of stage-specific embryonic antigen-3– positive (SSEA-3+) and SSEA-3– cells from hESC cultures. Although both SSEA-3+ and SSEA-3– cells could initiate pluripotent hESC cultures, we show that they possess distinct cell-cycle properties, clonogenic capacity and expression of ESC transcription factors. Our study provides formal evidence for heterogeneity among self-renewing pluripotent hESCs, illustrating that this isolation technique will be instrumental in further dissecting the biology of hESC lines.

hESCs are derived from the inner cell mass of pre-implantation embryos1 and are rigorously defined by two distinguishing properties: (i) ability to self-renew in culture over serial passage while (ii) retaining pluripotent developmental potential into all three primordial germ layers1,2. It is the combination of these defining characteristics that gives hESCs the unprecedented potential to act as a source of cells for cellular therapies in addition to allowing investigation of early events of human developmental biology. But before these potential applications of hESCs can be effectively pursued, a comprehensive understanding of the cellular relationships existing within hESC cultures and factors involved in the maintenance of hESC properties is required. hESCs are cultured either on feeder layers1,3–9 or in feeder-free conditions10–12. Xeno- and feeder-free hESC culture systems are essential for large-scale expansion of hESCs for future use in clinical applications10–12. Presently, however, basic understanding of hESC cultures is limited due to challenging aspects of hESC culture that have so far prevented application of certain available technologies that require selection of engineered hESCs, including the selection and expansion of rare populations. Selection of modified hESCs now is limited to inefficient methods that use manual selection of

colonies based on morphology and expression of a transgene either producing a fluorescent product or conferring drug resistance13–15. These methods are complicated by morphologic and phenotypic heterogeneity within hESC cultures that also contain differentiated populations10,16. As hierarchical arrangements among stem cell populations have been established in several systems, it is likely cellular hierarchies exist in hESC cultures, but has not been investigated because of the inability to effectively isolate subsets of clonogenic hESCs in an effective manner17–19. Accordingly, methods to isolate and subsequently characterize populations comprising hESC cultures would allow for a means to understand the nature of subsets and cellular interactions within hESC cultures. Here we describe a method that allows for feeder-free hESC culture regeneration after cell sorting. Seeding of isolated SSEA-3– expressing hESCs onto an irradiated, autologously derived hESCderived fibroblast-like cell (hdF) layer from the parent hESC line allows for rapid regeneration of individually sorted hESC based on transgene expression of fluorescent reporters and/or SSEA-3. The ability to clonally isolate subpopulations hESCs in a reproducible and quantitative manner allowed us to identify and characterize the presence of two subclasses of hESCs based on SSEA-3– and SSEA-3+ cells, revealing a previously unappreciated equilibrium of ESC subclasses that defines the hESC compartment and illustrating the instrumental role of this isolation technique to dissect hESC biology. RESULTS hESC cultures are heterogeneous Both morphological and phenotypic heterogeneity of hESCs cultured on feeder layers or under feeder-free conditions has been reported, with undifferentiated hESCs assumed to be restricted to dense clusters of cells (colonies) and apparently differentiated progeny, fibroblast-like cells, surrounding these colony structures10,20 (Supplementary Fig. 1 online). Fibroblast-like cells surrounding the putative hESC colonies, present in both feeder and feeder-free hESC culture systems (Supplementary Fig. 1), are generated by the colonies (data not shown) but do not express hESC markers (Fig. 1a). Immunocytochemical analysis of feeder-free hESC cultures demonstrated that hESC-associated

1McMaster Stem Cell and Cancer Research Institute, Hamilton, Ontario L9G 4L6, Canada and McMaster University, Faculty of Health Sciences, Department of Biochemistry, 1200 Main Street West, Hamilton, Ontario, Canada. 2The University of Western Ontario, Department of Microbiology and Immunology, 1151 Richmond Street, Suite 2, London, Ontario, Canada. Correspondence should be addressed to M.B. ([email protected]).

RECEIVED 15 MAY; ACCEPTED 18 AUGUST; PUBLISHED ONLINE 21 SEPTEMBER 2006; DOI:10.1038/NMETH939

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Figure 1 | hESC cultures are heterogeneous: comparison of SSEA-3– and SSEA-3+ subpopulations. (a) Immunocytochemistry analysis of Oct4 (green) and SSEA-3 (red). Nuclei were stained with DAPI (blue). Scale bars, 200 mm. (b) Representative flow cytometry analysis of surface SSEA-3 expression in hESC cultures. Inset, isotype control. Histogram represents live-gated cells. (c) Histogram representation of DNA content of hESCs gated on SSEA-3– or SSEA-3+ cell population. (d) Graphic representation of cell-cycle compartments on days 2–6 after passage (mean value of four passages). Error bars, s.d., n ¼ 4. (e) Flow cytometry analysis of BrdU incorporation versus DNA content on SSEA-3– and SSEA+ subsets. At day 3 and day 7 after passage, hESC cultures were pulsed with BrdU for 6 h and then collected for analysis. No BrdU, representative staining control.

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monitored expansion for 18 d. Using 20,000–30,000 SSEA-3+ cells, we did not observe any proliferation, illustrating that 100 matrigel alone was insufficient to promote 0 200 400 600 8001,000 0 200 400 600 8001,000 0 200 400 600 8001,000 0 200 400 600 8001,000 0 200 400 600 8001,000 DNA content (7-AAD) proliferation of single SSEA-3+ hESCs cultured in feeder-free systems (Fig. 2a). markers, SSEA-3 and Oct-4, are localized to cells within the As fibroblast-like cells surrounded putative hESC colonies in colonies but not the surrounding fibroblast-like cells (Fig. 1a). feeder or feeder-free hESC culture conditions (Supplementary Even within colonies, however, marker expression is heterogeneous, Fig. 1), we evaluated the functional support of these cells. We presenting an unexploited opportunity to determine the nature of generated cultures of these fibroblast-like cells directly from parent cells comprising hESC colonies20 (Fig. 1a). We selected SSEA-3 feeder-free hESC cultures by enzymatic digestion with collagenase expression to indicate undifferentiated hESCs based on coexpres- IV followed by manual removal of residual colonies (Fig. 2). We sion with Oct-4 within hESC colonies and its rapid downregulation termed these cells hESC-derived fibroblast-like cells (hdFs). Culupon hESC differentiation21,22, but it is unclear whether the SSEA- tures of hdFs can be maintained using hESC culture conditions for 3–expressing cells within the culture sustain colony formation and at least eight passages, and can be frozen and thawed successfully. are required for maintenance of hESC lines. Analysis of hdF cultures revealed a lack of SSEA-3 expression, Immunostaining revealed SSEA-3–expressing (SSEA-3+) and which supports the observation that SSEA-3 expression is restricted non–SSEA-3–expressing (SSEA-3–) populations within hESC cul- to cells within hESC colonies (Fig. 2c,d). tures (Fig. 1a,b). To further characterize these two populations, we We selected hESCs based on SSEA-3 expression and seeded them analyzed the cell-cycle patterns of SSEA-3– and SSEA-3+ cells within as single cells onto matrigel alone or matrigel plus a layer of hdFs H1 and H9 hESC cultures (Fig. 1c). Detailed analyses using mitotically inactivated with a sublethal dose of X-ray radiation propidium iodide staining23,24 over 5 d revealed that the SSEA-3– (ihdFs; Supplementary Fig. 2 online). We monitored the expanpopulation cycled less actively than the SSEA-3+ population sion of sorted hESCs by visual quantification of colony-like (Fig. 1d). Additionally, decreased BrdU incorporation (Fig. 1e) structures generated starting at day 4 post-sort (seeding of FACS and amounts of phosphorylated histone-3 (H3-P) (Supplemen- isolated cells is day 0). Regeneration of hESC cultures from sorted tary Fig. 1) in the SSEA-3– subset compared to the SSEA-3+ subset hESCs, determined by successful passage after either seeding onto confirmed the slower cell cycle of the SSEA-3– subset within hESC ihdFs or matrigel alone after the initial sort, was 100% (n ¼ 15, cultures. These observations suggest that the SSEA-3– and SSEA-3+ P ¼ 0.0007) in the presence of ihdF layers, in comparison to 33% cells may represent unique physiological states of cells within when hESCs were seeded onto matrigel (n ¼ 12). The presence of hESC cultures. any observable colonies on matrigel was due to a tenfold increase in cell number seeded (200,000 cells) versus 20,000 where no colony FACS isolation and expansion of subsets from hESC cultures formation could be observed upon seeding onto matrigel (Fig. 2a). To examine whether the SSEA-3+ population within hESC cultures This represents a threefold increase in the rate of establishment of contains undifferentiated hESCs responsible for sustaining hESC hESC lines from sorted cells compared to seeding onto matrigel lines, we directly isolated the SSEA-3+ population using alone. We also observed a significant 60-fold increase in the number fluorescence-activated cell sorting (FACS) from dissociated hESC of colonies generated per 103 cells seeded, in the presence of ihdF layers compared to seeding SSEA-3+ hESCs into wells coated with cultures stained for SSEA-3 (Fig. 2a). We used stringent sorting gates to select for viable hESCs expressing high levels of SSEA-3 matrigel alone (P ¼ 0.0115). The clonogenic efficiency (H1 hESCs, with reanalysis and restaining confirming 499% purity of the 1 in 380, n ¼ 7; H9 hESCs, 1 in 200, n ¼ 7; these frequencies were isolated subset (Fig. 2a). We seeded the resultant SSEA-3+ hESC not significantly different (0.08)) observed at day 5 with the use of subset onto matrigel-coated wells (as originally cultured) and ihdFs is equivalent to that previously observed for hESCs cultured BrdU


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on MEFs2,20 This translates to an average of four hESC colonies generated from 1,000 single cells from either H1 or H9 hESCs plated onto ihdFs. Health of ihdF layers diminished by 8–10 d post-irradiation, but the regenerated hESC colonies responded to the reduced support by producing fibroblast-like cells at the colony periphery. We passaged hESC colonies directly onto matrigel within 14 d, and the colonies did not exhibit any signs of feeder-layer dependence. In agreement with previous observations9, we were able to use hdFs derived from hESC lines different from the line being sorted, for example hdFs derived from H1-hESCs could be used to support sorted H9 hESCs, without any observable differences in colony formation, thus preventing the necessity of deriving hdF lines specific to each hESC line used. This method (i) allows for establishment of feeder-free hESC lines from clonogenic outgrowths and (ii) can use autologous sources derived from the hESCs. Generation of stably transduced hESCs using hdF support To determine whether the methods developed here could be used to rapidly generate stably transduced hESC lines, we selected hESC cultures resulting from low-efficiency, single-exposure transductions with lentivector containing eGFP or DsRed (Supplementary Fig. 3 online) based on fluorescent reporter and SSEA-3 expression (Fig. 3). We plated the resulting SSEA-3+ fluorescent reporter– expressing populations onto either ihdF layers or matrigel alone. Similar to the results observed for non-transduced hESCs, we observed a 2.5-fold increase in post-sort establishment of hESC lines for cells seeded onto ihdFs compared to matrigel alone (P ¼ 0.0331). Transduced hESC lines generated by this method demonstrated a significant sixfold increase in reporter expression (P ¼ 0.0023) over the original unselected hESC line (Fig. 3d–f). As per the nontransduced counterpart, these transduced hESC lines exhibit normal hESC marker expression (including SSEA-3, SSEA-4, Tra-1–60 and Tra-1–81) maintain a normal karyotype (data not shown) and are pluripotent, demonstrated by successful in vitro (Fig. 3g) and in vivo differentiation (Fig. 3h). Additionally, the resultant transduced hESC lines have maintained stable reporter expression in culture for over 1 year, with all colonies retaining reporter expression (data not shown). The methods

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Figure 2 | hESC-derived fibroblast-like cell (hdF) layers promote hESC culture regeneration from sorted hESCs. (a) Representative FACS plot of hESCs stained for SSEA-3 (left). Inset, isotype control. Purity analysis of SSEA-3+–gated hESCs (middle). FL1-H was the empty PMT channel that demonstrates lack of hESC autofluorescence. Phase contrast images of SSEA-3+–gated hESCs seeded onto matrigel alone after 6 d of culture showing different morphologies (right). Bottom right image shows higher magnification of the boxed area in the left image. Scale bars, 500 mm (left) and 200 mm (right). (b) Phase contrast image of a confluent day-9 hdF culture. Inset, hdF culture one day after passage. Scale bars, 500 mm. (c–d) Culture morphology and associated flow-cytometric analysis of SSEA-3 expression in hESC (c) and hdF (d) cultures. Scale bars, 500 mm. Insets: isotype controls. FL4-H was the empty PMT channel that demonstrates lack of hESC autofluorescence. The images in c and d show morphology of cultures on which flow cytometry was performed.

developed here to select for colony-forming hESCs and for the expansion of single cell populations can be used as an efficient and effective method to generate stably transduced hESC lines. hdF layers do not generate post-sort hESC colonies As hdFs were derived from hESC cultures, it was possible that the resultant colony growth was due to proliferation of a low level of colony-forming hESCs present in the hdF layers and not because of the proliferation of sorted hESCs seeded onto the ihdF layers. We observed no colony formation in ihdF cultures after 11 d at any of the irradiation doses tested or in maintained hdF cultures (data not shown). To confirm that the observed colonies were the result of proliferation of the sorted cells, we sorted eGFP-hESC and DsRedhESC lines, expanded them as described above (Fig. 3a), and analyzed the resulting colonies for fluorescent reporter expression. Fluorescent reporter expression was limited to all observed colonies and not the ihdFs, demonstrating that colonies generated on ihdF layers were the result of reporter-expressing SSEA-3+ sorted cells and not latent hESC colony formation from the ihdF layer (Fig. 3b,c). Additionally, we sorted the hESC line H9 (XX karyotype) and expanded it on ihdFs derived from hESC line H1; subsequent karyotypic analysis of the regenerated culture revealed an XX karyotype with no XY karyotypes detected, conclusively demonstrating that colonies were arising from the sorted cells and not the ihdFs. Clonal isolation reveals subclasses of self-renewing hESCs. To confirm our observations that the ability to regenerate hESC cultures was limited to the SSEA-3+ population, we selected and seeded equal numbers of SSEA-3– and SSEA-3+ cells onto ihdFs. Using FACS, we isolated SSEA-3– and SSEA-3+ cells from both H1 and H9 hESC cultures with 499.0% purity with identical results (Fig. 4a). In addition to application of stringent sort gates (Fig. 4a), restaining of both SSEA-3– and SSEA-3+ sorted populations demonstrated that the SSEA-3– population was still negative for SSEA-3 (99.75% ± 0.159 SSEA-3–, n ¼ 4), and that the SSEA-3+ subset did not contain SSEA-3– cells (97.5% ± 1.000 SSEA-3+, n ¼ 4), providing an overall indication that incomplete SSEA-3 staining did not occur. We monitored colony formation from single NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 809

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plated cells over 14 d after initial seeding (Fig. 4b,c), allowing comparison of clonogenic potential between the two subsets. As anticipated, the SSEA-3+ population yielded colonies (Fig. 4b) and resulted in regeneration of hESC cultures that were morphologically and phenotypically identical to the original parent hESC cultures from which we isolated the subset, expressing Tra-1–60 and Tra-1–81 and SSEA-3 at similar frequencies (Fig. 4d). Surprisingly, the SSEA-3– population also generated readily identifiable colonies (Fig. 4b) and cultures that ultimately contained cells expressing hESC markers initially at lower frequencies than cultures established using SSEA-3+ cells (Fig. 4d). The SSEA-3– population consistently generated 2–3-fold less colonies than the SSEA-3+ population, which is dramatically greater than the 4100-fold difference in the rate of colony formation expected if the resultant colonies observed in the SSEA-3– population were due to contaminating SSEA-3+ cells alone (given the 499.0% purity; Fig. 4a). The rate of colony formation from the SSEA-3– cells was decreased (Fig. 4c), further supporting the lack of SSEA-3+ in the seeded SSEA-3– fraction. Similar data were generated using H1 and H9 hESCs. Cultures generated from both SSEA-3– and SSEA-3+ cells were able to generate teratomas containing all three germ layers upon injection into immunodeficient mice (Supplementary Fig. 4 online), indicative of retained pluripotent potential and retained chromosomal stability (Supplementary Fig. 5 online). Clonogenic capacity of sorted hESCs is retained Despite the generation of colonies and establishment of cultures with morphology, phenotype and differentiation capacity similar 810 | VOL.3 NO.10 | OCTOBER 2006 | NATURE METHODS

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Figure 3 | Colonies arise from SSEA-3+–gated hESCs, not irradiated hdF layer. (a) FACS plots indicating reporter expression and SSEA-3 expression of sorted transduced hESC cultures. SSEA3+ reporter+ gate indicated. (b–c) Regeneration of transduced hESC cultures from SSEA-3+ reporter+ cells seeded onto ihdF layer. Phase contrast, fluorescence and overlaid images of GFPhESC (b) and DsRed-hESC (c) cultures at days 11 and 7 post-sort, respectively. Arrowheads indicate nontransduced, non–reporter-expressing ihdFs. Scale bars, 100 mm. (d-f) Selection of low-level transduced hESC cultures, based on coexpression of reporter and SSEA-3. Phase and corresponding fluorescent images of transduced hESCs, unsorted (top) and sorted based on reporter and SSEA-3, seeded onto ihdFs (bottom; d). Scale bars, 500 mm. Flow-cytometric analysis of GFP-hESC (left) and DsRed-hESC (right), before and after selection on SSEA-3 and reporter expression (e). Mean frequency of reporter expression by transduced hESCs before and after selection based on SSEA-3 and reporter expression (f). Error bars, ±s.e.m.; n ¼ 4. GFP: pre-sort 8.2 ± 0.4, post-sort 76.5 ± 2.5, *P ¼ 0.0309; DsRed: pre-sort 25.2 ± 1.5, postsort 84 ± 1.0, *P ¼ 0.0323. (g–h) In vitro and in vivo differentiation of SSEA-3+ sorted transduced hESCs. Fluorescence image of day 8 embryoid bodies generated from GFP-hESCs (left) DsRedhESCs (right; g) Scale bars, 500 mm. Fluorescence image of teratoma generated from GFP-hESCs, nuclei stained by DAPI (blue; h). Scale bar, 50 mm.

to hESCs, the ability of SSEA-3– or SSEA-3+ cultures to retain clonogenic capacity and/or inherit similar clonogenic properties as their parent subsets is unknown. To directly examine this functionally, we used a secondary replating approach of cells onto ihdFs, combined with reisolation of SSEA-3– and SSEA-3+ progeny derived from cultures established from either SSEA-3– or SSEA3+ parents. The experimental design is illustrated in Figure 4e. The clonogenic efficiency generated for both SSEA-3– and SSEA+ 3 populations were retained for each phenotype between primary and secondary isolations during serial replating (Fig. 4f). This was consistent regardless of whether the parent culture was established with SSEA-3– or SSEA-3+ subsets. Although we observed a slight decrease in clonogenic potential of the SSEA-3+ population isolated from the SSEA-3– parent culture, this was not significant and maybe due to insufficient time allowed to re-establish the equilibrium of SSEA-3– and SSEA-3+ hESCs within the SSEA-3– culture before the secondary sort. These results were identical using H1 and H9 hESCs. To further evaluate the unique properties of SSEA-3– and SSEA+ 3 cells from hESC cultures and continue to validate the single-cell origin of this clonogenic assay, we compared the functional potential of these two subsets by FACS-based single-cell deposition into individual 96-well plates containing ihdFs. The frequency of colony generation, regardless of whether the parent culture was established from SSEA-3– or SSEA-3+ subsets, was as observed in the bulk culture experiment (data not shown). These data provide support for the unique properties of SSEA-3– and SSEA-3+ colony forming cells, and indicate that these characteristics are not inherited by parent cultures, but retained and tightly associated

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with the cellular phenotype. This establishes that hESCs, defined by clonogenic self-renewing and pluripotent properties, are heterogeneous in nature. As clonogenic SSEA-3– hESCs give rise to both SSEA-3– and SSEA-3+ hESCs that have secondary clonogenic properties upon replating, the SSEA-3– and SSEA-3+ subsets appear to function in a previously unappreciated equilibrium to maintain the homeostasis of the hESC cultures. In addition to allowing for the efficient regeneration of hESC cultures from sorted populations, this method allows for the development of clonal assays for detection of single self-renewing hESCs. Bi-daily observation of selected reporter-expressing hESCs suggested that colonies arose as a result of proliferation of a single hESC rather than migration of the sorted hESCs together (data not shown). The possibility still exists that colonies could be seeded by more than one hESC, but 490% of the sorted hESCs seeded into the well did not attach, and careful observation of the attached cells on day 1 did not reveal the presence of doublets or clusters of hESCs. SSEA-3– and SSEA-3+ hESCs function autonomously To examine and define the relationship among the SSEA-3– and SSEA-3+ subsets during the initial phases of proliferation and

clonogenic growth, we reconstructed hESC cultures by coincubation of individual GFP- and DsRed-labeled SSEA-3– and SSEA-3+ cells. This approach permits interactions between the SSEA-3– and SSEA-3+ hESCs, while distinguishing the individual clonogenic behavior of SSEA-3– versus SSEA-3+ subsets. Initially, we independently examined the clonogenic potential of all four isolated populations and detected similar properties to unmarked SSEA3– and SSEA-3+ isolates (Fig. 4g). GFP+ or DsRed+ SSEA-3– cells cocultured with either GFP+ or DsRed+ SSEA-3+ cells had identical clonogenic potential to the individually cultured subsets (Fig. 4g). Colonies expressing both GFP and DsRed appeared at an incidence of 0.4% (7 out of 1,706), but these colonies were not mosaic (comprised of evenly distributed GFP+ and DsRed+ cells) suggesting that they arose from cells settling adjacent to each other and proliferating, rather than cells migrating together (Fig. 4h). This observation and the results of single-cell deposition experiments, in which single cells were sorted directly into individual wells (data not shown) provided direct evidence of the clonogenic origin of colonies detected using this assay system that depends on ihdF clonal support. These results reveal that clonogenic hESCs reside in two distinct states within the culture and further demonstrate that NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 811

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Figure 5 | SSEA-3– and SSEA-3+ sorted cells have distinct hESC marker expression. (a) Immunocytochemical analysis of SSEA-3– and SSEA-3+ cells 24 h after sorting. (b) Quantitative RT-PCR of Pou5f1 and Nanog mRNA expression in SSEA-3– and SSEA-3+ subsets immediately after isolation by FACS. Gapdh loading control. Graphs at the bottom show representative amplification curves. (c) In situ hybridization of Pou5f1 costained for SSEA3 antigen in hESC cultures; SSEA-3 (green), Pou5f1 transcript (red), DAPI (blue). Anti–SSEA-3, anti-sense Pou5f1 probe; arrow, SSEA-3+, POU5F1+; arrowhead: SSEA-3–, POU5F1+ (left). Controls: isotype control, no probe (top right); anti-SSEA-3, sense Pou5f1 probe (middle right); RNase A treatment before hybridization, anti–SSEA-3 and anti-sense Pou5f1 probe (bottom right). Scale bars, 50 mm (left, and middle and bottom right), 100 mm (top right). (d) Proposed model of hESC compartmentalization.

these two phenotypically compartmentalized states function independently to re-establish culture equilibrium. Distinct Oct-4 and Nanog profiles for SSEA-3– and SSEA-3+ cells To further characterize molecular differences that distinguish these pluripotent stem cell states, we used immunocytochemical analysis to examine the expression of markers associated with hESCs such as SSEA-3 and SSEA-4, and transcription factors Oct-4 and Nanog on the sorted populations within 24 h of isolation1,22,25–27. In addition to SSEA-3 expression, the SSEA-3+ subfraction expressed SSEA-4, Oct4 and Nanog (Fig. 5a). SSEA-3– cells, however, did not express any of these markers of undifferentiated cells (Fig. 5a), suggesting this subset represents a previously unidentified state of selfrenewing pluripotent hESCs with clonogenic ability. Quantitative real time PCR demonstrated that Pou5f1 (also known as Oct-4) and Nanog mRNA were equally expressed in SSEA-3– and SSEA-3+ subsets (Fig. 5b). Notably, although hdFs do not express SSEA-3, unlike sorted SSEA-3– cells, hdFs do not express Oct-4 or Nanog (Supplementary Fig. 6 online) and are functionally devoid of cells able to generate colonies containing cells expressing Oct-4, Nanog, 812 | VOL.3 NO.10 | OCTOBER 2006 | NATURE METHODS

SSEA-3, SSEA-4, Tra-1–81 or Tra-1–60. We performed in situ hybridization for Pou5f1 and costaining for SSEA-3 antigen in parent hESC cultures (Fig. 5c) to directly evaluate expression of Pou5f1 among SSEA-3– and SSEA-3+ cells within the hESC colonies. Nearly all SSEA-3+ cells co-express Pou5f1 transcript, whereas SSEA-3– cells residing within the hESC colony are only positive for Pou5f1 transcript (Fig. 5c). Based on the lack of correlation between Oct-4 and Nanog protein and mRNA in SSEA-3– cells, we suggest that a dynamic equilibrium is established between the SSEA-3– and SSEA-3+ hESC compartments by translational control of stem cell associated factors Nanog and Oct-4 from their putative transcripts. DISCUSSION We report here that specific populations within feeder-free hESC cultures can be isolated and expanded using FACS to select specific targeted subsets of hESCs, based on surface expression of SSEA-3 or combined expression of SSEA-3 and a fluorescent reporter in transduced hESCs. This technology uses a unique support layer of hdFs directly derived from undifferentiated


ARTICLES hESCs. This method provides a new alternative to enrich genetically modified hESCs and allows dissection of hESC culture heterogeneity10,22 by the selection and quantitative analysis of individual hESCs in a previously unavailable clonogenic assay. To support this protocol, we developed a method to efficiently isolate the fibroblast-like cells (hdFs) directly from hESC cultures to generate hdF layers, without the differentiation processes required to generate feeder layers from hESCs5–9. Although it may be possible to use hdFs as a general feeder layer for hESC culture, we present them here only as a temporary support layer that promotes regeneration of hESC cultures from FACS-selected feeder-free hESCs. The supportive characteristics of hdF layers, similar to the autologous human feeder lines that were previously derived9, are not restricted to the hESC line origin of the hdF, but hdFs are distinct from Diff Miz-hES6 cells9, as hdFs are derived directly from hESC cultures and maintained in hESC culture conditions. This may contribute to preventing feeder-layer dependence arising in the regenerated hESC lines. Our method using hdFs should not be not restricted to the two lines (H1 and H9) described in detail here, as hdFs have also been observed in H7 HES3 and HES4 hESC lines cultured in feeder-free systems (data not shown). As hdFs are present in hESC cultures and are produced by colonies generated from sorted cells, the regenerated hESC cultures do not develop dependence on the presence of hdFs. Based on differences in physical position, phenotype and inability to differentiate into mature lineages or form teratomas (data not shown), hdFs are distinct from hESC-derived cell types previously reported5–9, and hdF cultures do not spontaneously generate colonies or regenerate hESC cultures, making them distinct from the SSEA-3– hESCs coisolated from hESC cultures during selection based on SSEA-3 expression. Similar to the published observations and reports of stromal-like cells10,16,28, our laboratory continues to characterize the lineage and cell fate origin of hdFs. Although selection of hESCs based on a marker representative of the undifferentiated state has been demonstrated using hESCs cultured on MEFs, along with genetic modifications and selection, the pluripotency of hESC cultures resulting from these sorted cells was not addressed20,29. The method presented here demonstrates the ability to isolate populations within feeder-free hESC cultures and examine their self-renewal and differentiation potentials. With hESCs being defined by their self-renewal ability in culture and pluripotent developmental potential, our study reveals two states of hESCs distinguished initially by SSEA-3 expression, then by clonogenic capacity and amounts of Oct-4 and Nanog protein. Notably, less active SSEA-3– hESC do not initially express protein markers associated with hESCs, such as Nanog and Oct-4, further establishing a molecular distinction among these classes of hESCs. But the regeneration of SSEA-3– and SSEA-3+ hESCs in cultures seeded with their respective isolated counterparts demonstrates the dynamic equilibrium that exists between these two states of hESCs, further distinguished by levels of transcription factors Oct-4 and Nanog associated with hESC fate (Fig. 5d). This observation suggests that the present surrogate markers used to define and compare hESCs should be used with caution. The specific phenotype of SSEA-3– hESCs within the bulk SSEA-3– population isolated from hESC cultures has yet to be determined. Considering our experiments, it is highly unlikely that colonies generated from SSEA-3– isolated cells are the result of contaminating SSEA-3+ cells, based on sort purities, resulting ratios of colony formation between

the SSEA-3– and SSEA-3+ populations, different rates of colony formation, phenotypic differences and the overwhelming consistency of our results among individual experiments, and between hESC lines used. Based on our observations, we propose a model for hESC heterogeneity where hESCs reside in both less (SSEA-3–) and more (SSEA-3+) active states that comprise the human stem cell compartment (Fig. 5d). These observations are reminiscent of previous results from human teratocarcinoma cells30, suggestive of a linkage to hESC hierarchical arrangements and stem cell transformation. As present methods for the growth and differentiation of hESCs envision that hESCs represent a homogeneous stem cell population, our findings have direct impact on the future design and development of conditions used to control and investigate pluripotency and self-renewal. Ultimately, these findings suggest that further detailed analysis of cells and populations within hESC cultures is required to better understand the underlying biology of individual hESCs and how these relationships may participate and respond to directed lineage specification. The method presented here circumvents the limitations that thus far have prevented efficient clonal analysis of hESCs and provides a method that allows for hESC self-renewal to be quantitatively and comparatively examined. Our identification of phenotypically distinct classes of self-renewing and pluripotent hESCs directly demonstrates the utility of clonogenic assays and selection methods. METHODS hESC culture and embryoid body formation. We cultured hESC lines H9 and H1 (ref. 1) on matrigel in MEF-CM with 8 ng/ml basic fibroblast growth factor (bFGF)31 or serum replacement (SR)-bFGF medium with 36 ng/ml bFGF32. We maintained hESC lines as previously described31,32. Briefly, hESC colonies were dissociated with collagenase IV (Gibco) for 5 min and passed every 6–7 d. Occasionally, hESC colonies maintained in MEF-CM or SRbFGF were physically dissociated during passaging to reduce the numbers of fibroblast-like cells32. Both H1 and H9 hESC lines were used for analyses, and generated similar results and observations. Assessment of undifferentiated markers by flow cytometry. We dissociated hESCs10, stained them for Tra-1–60 (Chemicon), Tra1–81 (Chemicon) or SSEA-3 (Develop Studies Hybridoma Bank, mAb clone MC-631), and performed secondary dection with AlexaFluor-647–goat anti-mouse immunoglobulin G (IgG; Molecular Probes), goat anti-mouse IgG-PE or goat anti-mouse IgG-FITC (Immunotech). We identified live cells by 7-aminoactinomycin D (7-AAD) exclusion and analyzed them for surfacemarker expression using FACSCalibur (BDIS). We analyzed data using FlowJo software (Tree Star). hdF layers. We treated hESC cultures with a high proportion of fibroblast-like cells for 20 min with collagenase IV, then removed collagenase IV and rinsed the wells with MEFCM with 8 ng/ml bFGF or SR-bFGF to collect the fibroblast-like cells. We transferred supernatant containing the cells to fresh matrigel-coated wells. We changed the medium every other day and passaged hdFs when confluent in the same manner as for hESCs. We froze hdFs in hESC medium with 10% DMSO and thawed them successfully. One day before sorting, we collected hdFs from confluent hdF cultures by trypsin-EDTA treatment, dissociated them to single NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 813

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cells by trituration, filtered them through a 70-mm cell strainer, and subjected these hdFs at B2 105 cells/ml to 3788 Rad (Faxitron Cabinet X-ray System). We seeded 4–5 105 ihdFs per well in a 6-well tissue-culture plate coated with matrigel. We seeded non-irradiated hdFs at 4 104 cells per well onto matrigelcoated 6-well tissue-culture plates. See Supplementary Protocol 1 online for a detailed step-by-step protocol for derivation of hdFs. Sorting of hESCs and regeneration of sorted hESC cultures. We dissociated hESCs, transduced or untransduced, and stained them for SSEA-3 as described in Supplementary Methods online. We prepared single cells from hESCs as previously described10. Briefly, 70–80% confluent hESC cultures were subjected to collagenase IV for 15–20 min at 37 1C, followed by exposure to cell dissociation buffer (Invitrogen) for 10 min at 37 1C. We pelleted the cells, then triturated gently to manually dissociate them and passed them through a 70-mm cell strainer to remove remaining undissociated clumps. We identified live transduced and untransduced hESCs by 7-AAD exclusion and sorted the cells based on SSEA-3 expression alone or by co-expression of SSEA-3 and reporter gene, using a FACS Vantage SE (BD Biosciences; Fig. 2a). We collected sorted cells into a 5 ml FACS tube containing 0.5 ml of MEFCM with 8 ng/ml bFGF or 0.5 ml of SR-hES medium with 36 ng/ml bFGF, based on the medium used to grow the sorted hESCs. We centrifuged the sorted cells at 250g for 3–5 min and resuspended them in the appropriate hESC medium for seeding into wells of a 6-well tissue-culture plate containing matrigel alone, matrigel plus ihdFs or matrigel plus hdFs. We changed media daily, and monitored growth of colonies and scored them every other day until ready for passage or day 20 after seeding. See Supplementary Protocol 2 online for a detailed step-by-step protocol for clonal isolation of FACS-sorted cells onto hdFs. We passaged the resulting colonies as indicated above and analyzed them by FACS to determine presence of hESC markers and reporter expression for transduced hESCs. We generated teratomas and performed histological analysis as previously described32. Briefly, we injected approximately 4–8 104 undissociated GFP- and DsRed-hESCs intratesticularly into male nonobese diabetic severe combined immunodeficient (NOD-SCID) mice. We killed the mice at 8 weeks and removed the tumors. We embedded tumors in frozen tissue-embedding gel (Fisher), snapfroze them in liquid nitrogen and stored them at –80 1C. We examined cryostat sections (5 mm) histologically after hematoxylin-and-eosin staining. Statistical analysis. All error bars represent ± s.e.m. Two-tailed paired or unpaired t-tests were performed, as appropriate. Additional methods. Descriptions of the analysis of Oct4 and SSEA-3 expression in hESC cultures by immunocytochemistry, DNA content analysis, BrdU labeling and incorporation, analysis of histone-3–P, production of lentivirus, transduction of hESC with lentivirus, reverse transcription and PCR, as well as in situ hybridization are available in Supplementary Methods. Note: Supplementary information is available on the Nature Methods website. ACKNOWLEDGMENTS We acknowledge R. Mondeh, A. Rouleau and J. Yang for outstanding technical assistance, and D. Sheerar for cell isolation. We thank K. Vijayaragavan for critical

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review of the manuscript. This research was funded by a grant from the Canadian Institutes of Health Research (CIHR) and the National Centres of Excellence–Stem Cell Network Program and postgraduate scholarship award from the Stem Cell Network to M.H.S., and CIHR Canadian Graduate Scholarships to S.C.B. and K.C. AUTHOR CONTRIBUTIONS M.H.S. performed experiments and assisted in writing the paper; M.B., K.C., P.M. and S.C.B. assisted with experiments; M.B. wrote and edited the paper. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/naturemethods/ Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ 1. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998). 2. Amit, M. et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271–278 (2000). 3. Genbacev, O. et al. Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders. Fertil. Steril. 83, 1517–1529 (2005). 4. Lee, J.B. et al. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol. Reprod. 72, 42–49 (2005). 5. Xu, C. et al. Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22, 972–980 (2004). 6. Stojkovic, P. et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23, 306–314 (2005). 7. Wang, Q. et al. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells 23, 1221–1227 (2005). 8. Lee, J.B. et al. Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 128, 727–735 (2004). 9. Yoo, S.J. et al. Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells. Exp. Mol. Med. 37, 399–407 (2005). 10. Xu, C. et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971–974 (2001). 11. Li, Y., Powell, S., Brunette, E., Lebkowski, J. & Mandalam, R. Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol. Bioeng. 91, 688–698 (2005). 12. Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–845 (2004). 13. Ma, Y., Ramezani, A., Lewis, R., Hawley, R.G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21, 111–117 (2003). 14. Costa, M. et al. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat. Methods 2, 259–260 (2005). 15. Vallier, L. et al. Enhancing and diminishing gene function in human embryonic stem cells. Stem Cells 22, 2–11 (2004). 16. Carpenter, M.K. et al. Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev. Dyn. 229, 243–258 (2004). 17. Mazurier, F., Gan, O.I., McKenzie, J.L., Doedens, M. & Dick, J.E. Lentivectormediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment. Blood 103, 545–552 (2004). 18. Evans, M.J. & Kaufman, M.H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981). 19. Hope, K.J., Jin, L. & Dick, J.E. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat. Immunol. 5, 738–743 (2004). 20. Enver, T. et al. Cellular differentiation hierarchies in normal and cultureadapted human embryonic stem cells. Hum. Mol. Genet. 14, 3129–3140 (2005). 21. Kannagi, R. et al. Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. EMBO J. 2, 2355–2361 (1983). 22. Draper, J.S., Pigott, C., Thomson, J.A. & Andrews, P.W. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J. Anat. 200, 249–258 (2002).


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28. Rosler, E.S. et al. Long-term culture of human embryonic stem cells in feeder-free conditions. Dev. Dyn. 229, 259–274 (2004). 29. Eiges, R. et al. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr. Biol. 11, 514–518 (2001). 30. Andrews, P.W. et al. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor. Differentiation 29, 127–135 (1985). 31. Chadwick, K. et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 102, 906–915 (2003). 32. Wang, L., Li, L., Menendez, P., Cerdan, C. & Bhatia, M. Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105, 4598–4603 (2005).

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