Niche-Dependent Gene Expression Profile of Intratumoral Heterogeneous Ovarian Cancer Stem Cell Populations

Intratumoral heterogeneity challenges existing paradigms for anti-cancer therapy. We have previously demonstrated that the human embryonic stem cells (hESC)-derived cellular microenvironment in immunocompromised mice, enables functional distinction of heterogeneous tumor cells, including cells which do not grow into a tumor in a conventional direct tumor xenograft platform. We have identified and characterized six cancer cell subpopulations each clonally expanded from a single cell, derived from human ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. These cancer cell subpopulations, characterized as cancer stem cell subpopulations, faithfully recapitulate the full spectrum of histological phenotypic heterogeneity known for human ovarian clear cell carcinoma. Each of the six subpopulations displays a different level of morphologic and tumorigenic differentiation wherein growth in the hESC-derived microenvironment favors growth of CD44+/aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44-/aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal markers and CD44 expression. In the current study, we delineate the distinct gene expression and epigenetic profiles of two such subpopulations, representing extremes of phenotypic heterogeneity in terms of niche-dependent self-renewal and tumorigenic differentiation. By combining Gene Set Enrichment, Gene Ontology and Pathway-focused array analyses with methylation status, we propose a suite of robust differences in tumor self-renewal and differentiation pathways that underlie the striking intratumoral phenotypic heterogeneity which characterize this and other solid tumor malignancies.     

The establishment of two rat colonic carcinomas in tissue culture. A basic prerequisite for standardized experiments on the biology of colonic carcinomas in vivo

Two rat colonic carcinomas (DMH-Co-1 and DMH-Co-2) derived from dimethyl-hydrazine-induced metastasizing adenocarcinomas were established as permanent cell lines. By means of electron microscopy, immunofluorescence microscopy and biochemical analysis of cytoskeletal components, it has been shown that both tumor cell lines retain in vitro the phenotypic characteristics of the primary tumors. The in vitro growth properties revealed only minor differences between the two cell lines. After retransplantation in vivo, DMH-Co-2 gave rise to moderately differentiated adenocarcinomas, whereas the tumors arising from DMH-Co-1 exhibited a continuum of differentiation encompassing adenocarcinomas, undifferentiated carcinomas and squamous cell carcinomas. These permanent cell lines offer the opportunity for isolating divergent subpopulations by in vitro cloning and facilitate standardized experiments on their biological behaviour in vivo.     

FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen

Salmonella can cause a typhoid fever like disease in genetically susceptible mice. In contrast to in vitro cell culture models, most host cells in infected tissues contain only one or two Salmonella, but a small subpopulation of infected host cells contains many Salmonella. It has been proposed that these various subpopulations have differential relevance during infection but to test this, methods for isolating such Salmonella will be required. We developed a method to purify differentially infected host cells by flow cytometry using Salmonella expressing the green fluorescent protein (GFP). Critical parameters for this method were sufficient GFP expression to detect infected cells against strong host cell autofluorescence, and low variation in GFP content of individual Salmonella. We evaluated more than hundred different GFP-constructs but only one single-copy chromosomal P(sifB)-gfp fusion met these criteria and enabled differentiation of weakly and highly infected cells based on total GFP fluorescence. Confocal microscopy of sorted cells confirmed the successful separation of these subpopulations. In addition to isolation of infected cells, our method also enabled enumeration of the subpopulations and phenotypic characterization by staining with antibodies to surface markers. Surprisingly, a small subpopulation of highly infected host cells contained the majority of Salmonella but based on MHC II and ICAM I expression, this subpopulation was not more strongly activated than weakly infected cells. Our method will facilitate future characterization of the different subpopulations and the identification of bacterial and host factors that control Salmonella load and proliferation in vivo.     

Terminally differentiated postmitotic tumor cells in a rat rhabdomyosarcoma cell line

A permanent rat rhabdomyosarcoma cell line (BA-HAN-1C) has been established, the phenotype of which is characterized by the coexistence of undifferentiated mononuclear cells and differentiated multinuclear myotube-like giant cells. The failure of attempts to separate these two cell types by repeated recloning procedures indicates their close histogenetic relationship and suggests that differentiation in this tumor proceeds in a similar manner to that in normal striated muscle where postmitotic myotubes arise from mononuclear myoblasts by fusion. The morphologically undifferentiated mononuclear tumor cells were shown to be actively proliferating and to incorporate thymidine methyl-3H(3H-TdR). The myotube-like giant cells neither incorporated 3H-TdR nor underwent mitosis or exhibited any clonogenic potential. After retransplantation into syngenic rats, tumor growth was markedly retarded when the tumor cell inoculum contained a high percentage of myotube-like giant cells. These data show that proliferative activity in this rhabdomyosarcoma cell line is confined to the mononuclear tumor cell compartment, the multinuclear myotube-like giant cells having withdrawn from the cell cycle and represent terminally differentiated postmitotic cells. This cell line should provide a valuable tool for further investigation of coherent aspects of proliferation and differentiation using various differentiation inducers.     

Initial GABAergic expression in embryonic amphibian neuroblasts after neural induction

At the late gastrula-early neurula stage some embryonic neuroblasts from neural plate and neural fold present apparently as a consequence of neural induction, the capability to develop in vitro into different neuronal subpopulations (cholinergic, dopaminergic, noradrenergic, somatostatinergic and some other peptidergic subpopulations without ongoing influences from the chordamesoderm (Duprat et al., 1987). Using the same in vitro model system, the aim of the present work was to delineate the abilities of these neuroblasts to develop GABAergic traits. The initial appearance and development of GABAergic phenotype has been quantitated by assaying the activity of glutamic acid decarboxylase (GAD). GAD activity was undetectable at the early gastrula stage (stage 8a) and was slightly measurable at the early neurula stage (stage 14- onset of the culture). It increased subsequently over the next 14 days in vitro. The temporal pattern of appearance and development of GAD activity in culture was in agreement with that observed in vivo. Immunocytochemical studies showed that GABA-like immunoreactivity was expressed in vitro in a subpopulation of neurons. Thus the developmental program for GAD expression and GABA phenotype maturation is acquired at least in some neuronal precursors. These data together with previously reported results on the expression of cholinergic, catecholaminergic and peptidergic phenotypes demonstrate that different neuronal subpopulations emerge near the end of gastrulation i.e. immediately after neural induction. The embryonic origin of this neuroblast heterogeneity remains to be determined.     

Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.     

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome

Application of stem cell biology to breast cancer research has been limited by the lack of simple methods for identification and isolation of normal and malignant stem cells. Utilizing in vitro and in vivo experimental systems, we show that normal and cancer human mammary epithelial cells with increased aldehyde dehydrogenase activity (ALDH) have stem/progenitor properties. These cells contain the subpopulation of normal breast epithelium with the broadest lineage differentiation potential and greatest growth capacity in a xenotransplant model. In breast carcinomas, high ALDH activity identifies the tumorigenic cell fraction, capable of self-renewal and of generating tumors that recapitulate the heterogeneity of the parental tumor. In a series of 577 breast carcinomas, expression of ALDH1 detected by immunostaining correlated with poor prognosis. These findings offer an important new tool for the study of normal and malignant breast stem cells and facilitate the clinical application of stem cell concepts.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.     

Derivation, propagation and differentiation of human embryonic stem cells

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.     

Asynchronism of thymocyte development in vivo and in vitro

Although fetal thymus organ culture (FTOC) has become widely used to investigate T-cell development, the differences between thymocyte development in vivo and in vitro (in FTOC) remain largely unknown. In this study, the viability and numbers of thymocytes recovered from embryonic thymus lobes in different gestation days (gd) mice or from 15 day embryonic thymus lobes cultured for different days in FTOC system were evaluated. The expression of CD3, CD4, CD8, CD95 ligand (CD95L), and CD69 on thymocytes were analyzed by FACS. The results showed that thymocytes, either in vivo or in vitro, could differentiate from double negative (DN) cells to double positive (DP) cells and to single positive (SP) cells. But the number of total thymocytes and the percentage of DP cells in vitro were less than that in vivo, and the expression of CD95L and CD69 on thymocytes in vitro was higher than that in vivo. Our results suggested that although thymocyte development in vitro could recapitulate thymic development in vivo, the proliferation of thymocytes in vitro was less intensive than that in vivo; the differentiation of thymocytes in vitro was delayed compared with that in vivo; and the apoptosis and activation of thymocytes in vitro were higher than that in vivo. In conclusion, FTOC is a useful system for the study of T cell differentiation, but it is necessary to interpret the results from in vitro studies carefully since the thymocyte development in vitro is asynchronous from that in vivo.     

Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.     

Antibody-dependent cellular cytotoxicity effector cells lack Ia antigens.

Surface immunoglobulin (sIg)-positive and sIg-negative subpopulations of macrophage-depleted murine splenic lymphocytes were obtained by Sephadex anti-Fab immunoabsorbent fractionation. These lymphocyte subpopulations were analyzed for the presence of Thy 1 and Ia alloantigens and also for Fc receptors by fluorescence microscopy. Concurrently, these lymphocyte subpopulations were studied for effector cell activity in antibody-dependent cellular cytotoxicity (ADCC). Effector cells mediating ADCC were contained in the sIg-negative lymphocyte subpopulation and sIg-positive lymphocytes did not mediate cytotoxicity. The majority of sIg-positive lymphocytes were found to bear Ia antigens and Fc receptors, and these cell surface structures were associated in that treatment of these cells with anti-Ia sera inhibited binding of complexed immunoglobulin to Fc receptors. In contrast, most sIg-negative, Thy 1-negative lymphocytes lacked Ia Antigens, and the Fc receptors detected on such cells were not blocked by anti-Ia sera. In addition, a small subpopulation of sIg-negative, Ia antigen-positive, Fc receptor-positive lymphocytes was found. Elimination of this subpopulation of Ia antigen-positive cells from sIg-negative lymphocytes, by treatment with anti-Ia serum and complement, did not diminish ADCC effector cell activity in the resultant cell population when compared with untreated sIg-negative lymphocytes. Thus, in murine spleen, nonphagocytic mononuclear cells that lack both sIg and Ia antigens were shown to mediate ADCC.     

Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures

We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo. Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers. Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons. After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones. These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy. Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V. Johnson, 1983, J. Comp. Neurol., 221:31-41; and Blanks, J.C., and L.V. Johnson, 1984, Invest. Ophthalmol. Visual Sci., 25:546-557). This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo. Outer segments are not present at this stage of development either in vivo or in vitro. This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.     

Podocytes in metanephric organ culture express characteristic in vivo phenotypes

 Podocytes outgrown from isolated glomeruli in vitro have failed to express fully differentiated in vivo phenotypes. In an attempt to determine whether podocytes in metanephric culture accomplish terminal differentiation, as observed in vivo, we investigated expression of their characteristic phenotypic features in rat metanephric organ cultures using immunohistochemistry and electron microscopy. Rat metanephroi were harvested on embryonic day 12.5 and cultured on transmembrane filters for 9 days. Morphological examination revealed two maturation stages when the podocytes resembled those of the S-shaped body stage and maturational stages of glomeruli in vivo. Electron microscopy revealed that, firstly podocytes lost their intercellular contacts and, simultaneously, the tight junctions shifted into close proximity to cell bases, followed by foot process development. Immunohistochemistry demonstrated that the tight junction protein, ZO-1, and specific podocytic markers, pp44, 5-1–6, podocalyxin and vimentin were expressed in a cell maturity-dependent manner, as observed in newborn rat kidneys. Furthermore, glomerular basement membrane components, collagen type IV and laminin, were expressed in the glomerular center. Our findings that cell maturity-dependent expression of structural and functional phenotypes in podocytes in metanephric culture was the same as that observed in developing kidneys in vivo indicate that podocyte differentiation during glomerulogenesis may be operated by an intrinsic property, such as programmed cell fate. Furthermore, these highly differentiated podocytes in vitro may provide clues that will help to establish a podocyte culture system. Accepted: 26 February 1997     

Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress

Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS-Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.