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CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY
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The Esophageal Squamous Epithelial Cell—Still a Reasonable Candidate for the Barrett's Esophagus Cell of Origin?
Barrett's esophagus is the metaplastic change of the squa-mous epithelium lining the distal esophagus into an intestinalized columnar epithelium that predisposes to esophageal adenocarcinoma development. The cell that gives rise to Barrett's esophagus has not been identified definitively, although several sources for the Barrett's esophagus cell of origin have been postulated. One possible source is a fully differentiated squamous epithelial cell or a squamous epithelial progenitor or stem cell native to the esophagus that, through molecular reprogramming, either transdifferentiation or transcommitment, could give rise to an intestinalized columnar cell. Multilayered epithelium found in human beings and rodents with Barrett's esophagus and direct phenotypic conversion of mouse embryonic esophageal epithelium provide support for this. Limitations in current experimental approaches may explain why it has been difficult to fully change an esophageal squamous epithelial cell into an intestinalized columnar cell in vitro. (Cell Mol Gastroenterol Hepatol 2017;u:u-u; http:// dx.doi.org/10.1016/j.jcmgh.2017.01.015)
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Barrett's esophagus is the metaplasia in which a columnar epithelium with intestinal features and characterized by the presence of goblet cells replaces the normal stratified squamous epithelium lining the distal esophagus.1 This condition is important clinically because it increases the risk for developing esophageal adenocarci-noma.1 Barrett's esophagus is thought to occur secondarily to chronic epithelial injury and accompanying inflammation caused by gastroesophageal reflux. Despite intense research efforts, the molecular mechanisms underlying this meta-plastic change in epithelial phenotype have not been elucidated completely. Furthermore, the identity of the cell that gives rise to Barrett's esophagus has not been identified Q5 definitively. Identifying this "cell of origin" is essential because it has implications both for pathogenesis and treatment, especially in the setting of recurrent Barrett's esophagus after endoscopic ablation therapy.1
There are several postulated sources for the cell of origin in Barrett's esophagus.1 An early hypothesis of how Barrett's esophagus forms was that damaged squamous epithelium was simply replaced by proximally migrating columnar epithelial cells from either the squamocolumnar junction or gastric cardia. When gastroesophageal reflux was induced surgically in dogs and metaplastic columnar epithelium subsequently was found in an area denuded of epithelium above a residual squamous epithelial barrier, focus shifted to identifying a cell of origin native to the esophagus.2 Given that the normal epithelium found in the human esophagus is predominantly squamous (the
exception being the epithelium lining submucosal gland ducts and comprising the submucosal glands), 2 distinct hypotheses developed on how a squamous epithelial cell could give rise to a columnar epithelial cell. First, a fully differentiated squamous epithelial cell could undergo irreversible direct phenotypic conversion through molecular reprogramming into an intestinalized columnar cell without undergoing mitosis, a process termed transdifferentiation. Alternatively, a squamous epithelial precursor or stem cell could undergo molecular reprogramming leading to a change in the cell fate of progeny cells, a process termed transcommitment. The other potential source for the Barrett's esophagus cell of origin besides a proximally migrating columnar epithelial cell, a native squamous epithelial cell, or a native epithelial cell from an esophageal submucosal gland or duct, is an external circulating stem cell (ie, from the bone marrow).
Evidence for transdifferentiation or transcommitment of a squamous cell comes from studying tissue obtained from human patients with Barrett's esophagus and from rats that develop esophageal columnar metaplasia after the surgical induction of gastroesophageal reflux, and observations made during normal mouse esophageal development. In human patients, identification of a distinctive transition zone cell at the junction of squamous epithelium and Barrett's epithelium was reported by Shields et al.3 By scanning electron microscopy, these cells had ultrastructural features of both squamous and columnar epithelial cells. For example, they showed intercellular ridges, a characteristic feature of squamous cells, and short microvilli and bulging mucus, a characteristic feature of secretory columnar cells. Importantly, these cells clearly were different from Barrett's epithelial cells and normal gastroesophageal junction cells. By light microscopy, the junction of squamous and columnar epithelium in patients with gastroesophageal reflux disease often showed a multilayered epithelium with mucus-producing columnar cells overlying immature squamous cells. Further studies showed that basal cells in multilayered epithelium simultaneously expressed columnar cytokeratin 19 and squamous cytokeratin 4.4 Interestingly, multilayered epithelium also was observed in rats that had undergone a surgical procedure to induce bile reflux. In those rats that developed Barrett's esophagus in this setting, a multilayered epithelium was observed both at the neosquamocolumnar junction as well as in the midesophagus.5 The finding of multilayered epithelium at the junction of squamous and columnar epithelium is consistent with multilayered epithelium, representing an intermediate stage between squamous and Barrett's epithelium. Furthermore, because
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rats do not possess submucosal glands, multilayered epithelium, especially in the midesophagus, would appear to arise from a native esophageal squamous epithelial cell.
Similar to human beings, the mouse embryonic esophagus initially is lined by columnar epithelium that undergoes stratification and squamous differentiation during embryonic development. Initially, esophageal epithelial cells express the columnar cytokeratins 8 and 18. As development progresses, the expression of cytokeratins 8 and 18 diminish while basal squamous epithelial cells begin to express the squamous cytokeratin 14. Investigators from the Tosh laboratory developed an explant culture system to study this process more closely.6 Esophagi isolated from day 11.5 mouse embryos and grown in this culture system mimicked esophageal epithelial development observed in vivo. By using immunostaining for cytokeratin 8 and a cytokeratin 14-green fluorescent protein reporter, these investigators found that as esophageal development progressed, individual esophageal epithelial cells expressing cytokeratin 8 began simultaneously to express green fluorescent protein, or cytokeratin 14. This occurred even in the presence of inhibitors of apoptosis or cell division and ended with epigenetic silencing of cytokeratin 8 by promoter methylation. These results showed that an individual esophageal epithelial cell could undergo a direct phenotypic conversion from columnar to squamous. Reversing this process theoretically could lead to a squamous cell giving rise to a Barrett's esophagus-like phenotype.
The difference between transdifferentiation and trans-commitment depends on the differentiation status of the cell
A Mouse/Rat Esophagus
Keratin Layer
Epithelium 4-5 Cells Thick
Fibroblasts
Muscle
of origin. Multiple studies have identified discrete cell 176
populations from the mouse esophagus that appear to have 177
progenitor cell properties such as the ability to form col- 178
onies, give rise to organoids, and repopulate a fully differ- 179
entiated esophageal epithelium after injury. Various 180
markers to identify these cells include the exclusion of 181
Hoescht dye, Sca-1 positivity, Thy-1 positivity, the ability to 182
retain bromodeoxyuridine or tritiated thymidine, and the 183
expression of a6 integrin, b4 integrin, CD71, and/or CD73 184
(reviewed by Wang and Souza1). Investigators from the 185 Jones laboratory recently found that although mouse Q7186
esophageal epithelium contained squamous progenitor cells 187
that were functionally equivalent, quiescent label-retaining 188
stem cells were not present.7 Although mouse esophageal 189
epithelium is keratinized and uniformly 4-5 cell layers 190
thick, human esophageal epithelium is nonkeratinized, is 191
interrupted by slender folds of stromal papillae, and typi- 192
cally is much thicker than mouse esophageal epithelium 193
(Figure 1). Because of the papillae, human esophageal 194
epithelium can be divided into portions overlying stromal 195
papillae or portions overlying interpapillary regions. 196
Various groups using different techniques have reported 197
conflicting characteristics of these regions in regards to the 198
proliferative and stem cell compartments (reviewed by 199 Wang and Souza1). Although all agreed that the basal cells Q8 200
are the most proliferative, they disagreed as to whether the 201 basal cells overlying the papillae or those found in the interpapillary regions undergo asymmetric vs symmetric division, retain iodo-deoxyuridine or tritiated thymidine, or give rise to Ki-67-expressing proliferating cells. More
Human Esophagus
No Keratin
Epithelium ~25 Cells Thick
Interpapillary Region
Fibroblasts
Muscle
Stromal Papillae
Stromal Papillae
- SMG Duct
Submucosal Gland (SMG)
Figure 1. Schematic representation of the histologic structure of the mouse/rat and human esophagus. (A) The mouse/ rat esophageal epithelium is keratinized stratified squamous and comprises 4-5 cell layers. Fibroblasts and muscle are located deep to the epithelium. Submucosal glands are absent. (B) The human esophageal epithelium is nonkeratinized stratified squamous and comprises many cell layers. Stromal papillae divide the epithelium into regions overlying papillae and interpapillary regions. Secretions made by submucosal glands are carried by ducts, lined by cuboidal cells (shown in green), and released into the esophageal lumen. Fibroblasts and muscle are located deep to the epithelium. Created by Medical Media, Dallas Veterans Affairs Medical Center.
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■ 2017
235 Q9 recently, investigators from the Fitzgerald laboratory sorted
236 human esophageal epithelium from esophagectomy speci-
237 mens using antibodies against CD34 (to mark basal cells) 238Q10 and epithelial cell adhesion molecules (to mark suprabasal
239 cells) into 4 separate fractions.8 In colony-forming assays
240 and 3-dimensional (3D) organotypic cultures, all 4 fractions
241 of cells had similar characteristics, leading to the conclusion
242 that proliferative cells were widespread throughout the
243 human esophageal epithelium.
244 Although the identity of stem cells in the human
245 esophageal squamous epithelium continues to be debated,
246 most agree that the Barrett's esophagus cell of origin must
247 undergo some type of phenotypic change to acquire the
248 characteristics of intestinal differentiation. In vitro exper-
249 iments using differentiated human esophageal squamous
250 epithelial cells showed that they can undergo molecular
251 reprogramming. Treatment with acidified media and/or
252 bile salts, mimicking gastroesophageal reflux conditions,
253 led to down-regulation of squamous transcription factors
254 (eg, DNp63), and up-regulation of columnar (eg, SOX9) and
255 intestinal (eg, CDX1, CDX2, and FOXA2) transcription fac-
256 tors, as well as alterations in various signaling pathways
257 (reviewed by Wang and Souza1). These transcription fac-
258 tors are classified as such because they are expressed by
259 squamous, columnar, and intestinal mucus-producing
260 epithelial cells and have been shown to regulate markers
261 of squamous, columnar, and intestinal mucus-producing
262 differentiation. For example, the squamous transcription
263 factor DNp63 up-regulated expression of the squamous
264 cytokeratins 5 and 14, the columnar transcription factor
265 SOX9 induced expression of columnar cytokeratins 8 and
266 18, and the intestinal transcription factors CDX1 and CDX2
267 and the mucus transcription factor FOXA2 induced
268 expression of the intestinal mucin MUC2 in immortalized
269 human esophageal squamous epithelial cells (reviewed by
270 Wang and Souza1). Although many studies have depended
271 on expression analyses to show a phenotypic change, novel
272 3D organotypic culture systems and electron microscopy
273 have shown changes in cellular morphology after molecu-
274 lar reprogramming of human esophageal squamous
275 epithelial cells, especially when multiple genetic alterations
276 are induced simultaneously. For example, investigators 277Q11 from the Rustgi laboratory combined MYC and CDX1
278 overexpression with Notch pathway inhibition in the
279 telomerase-immortalized human esophageal squamous
280 epithelial cell line EPC2.9 This led to down-regulated
281 expression of squamous cytokeratins and up-regulated
282 expression of columnar cytokeratins and mucins. More
283 importantly, in 3D organotypic cultures, basal cells with
284 these 3 genetic alterations appeared morphologically
285 different and more elongated as shown by light and elec-
286 tron microscopy.
287 In summary, is the esophageal squamous epithelial cell
288 still a reasonable candidate for the Barrett's esophagus cell
289 of origin? Enthusiasm recently has shifted toward proxi-
290 mally migrating columnar cells from the squamocolumnar
291 junction or gastric cardia based on intriguing data from
292 genetic mouse models as well as toward submucosal glands
293 and their ducts based on lineage tracing with P53 and P16
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point mutations in human tissue specimens (reviewed by 294
Wang and Souza1). However, the presence of epithelial cells 295
that simultaneously express both squamous and columnar 296
cytokeratins in vivo in both the human and rodent esoph- 297
agus in the setting of gastroesophageal reflux suggests an 298
initial squamous source for Barrett's esophagus, if multi- 299
layered epithelium truly represents an intermediate stage 300
between squamous and Barrett's epithelium. In addition, the 301
presence of multilayered epithelium in the midesophagus of 302
rats, which do not have esophageal submucosal glands, after 303
reflux-inducing surgery argues strongly against submucosal 304
glands, their ducts, or a proximally migrating columnar cell 305
as a source of the multilayered epithelium.5 306
If an esophageal squamous epithelial cell remains as a 307
strong candidate for the Barrett's esophagus cell of origin, 308
the next question is why an esophageal squamous epithelial 309
cell has yet to be changed into an intestinalized goblet cell 310
in vitro. This is a difficult question to answer but likely is 311
owing to limitations in our current experimental ap- 312
proaches. First, we may not be using the correct esophageal 313
squamous cell as a substrate for transdifferentiation or 314
transcommitment experiments. Almost all studies in human 315
cell lines have been performed in differentiated, immortal- 316
ized cell lines, or in proliferative primary cell lines. Perhaps 317
these cell lines do not contain the requisite squamous pro- 318
genitor or stem cell with the plasticity to become an 319
intestinalized columnar cell. Organoid cultures of esopha- 320
geal squamous epithelium freshly isolated from patients 321
may allow genetic manipulation of cells with the required 322
plasticity. Second, phenotype switching from squamous to 323 intestinalized columnar may require multiple genetic alter-Q12324
ations in a specific combination and sequence. To date, the 325
majority of studies have examined the effects of altering the 326
expression of a single gene. A more logical approach 327
perhaps is to stably express a columnar transcription factor, 328
followed by an intestinal transcription factor, followed by a 329
mucus-related transcription factor. Based on metaplasia in 330
the pancreas where structural components have to be 331
down-regulated as well as up-regulated, down-regulation of 332
squamous genes also may need to be incorporated into this 333
sequence.10 Third, proper culture conditions for cells to 334
undergo transdifferentiation or transcommitment may be 335
underutilized. Novel culture systems with an air-liquid 336
interface and fibroblasts to permit epithelial-stromal in- 337
teractions, such as 3D organotypic culture or in vivo 338
transplant culture using a scaffold such as a denuded rat 339
trachea, might be required to induce recognizable 340
morphologic features of squamous or columnar differenti- 341
ation, or even gland formation. 342
Finally, while renewing our focus on esophageal squa- 343
mous epithelial cells as a potential source for the Barrett's 344
esophagus cell of origin, we should not ignore novel insights 345
gained from ongoing studies examining proximally 346
migrating columnar cells in genetic mouse models as well as 347
cells derived from esophageal submucosal glands and their 348
ducts. Perhaps each of these cells eventually may be shown 349
to be the source of the Barrett's esophagus cell of origin in 350
different patients. None of them have been disproved or 351
proved convincingly to date. 352
Cellular and Molecular Gastroenterology and Hepatology Vol. ■, No.
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DAVID H. WANG, MD, PhD Esophageal Diseases Center
Department of Internal Medicine and the Simmons
Comprehensive Cancer Center
University of Texas Southwestern Medical Center
Dallas, Texas
Medical Service
Dallas VA Medical Center
Dallas, Texas
References
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2. Gillen P, Keeling P, Byrne PJ, et al. Experimental columnar metaplasia in the canine oesophagus. Br J Surg 1988;75:113-115.
3. Shields HM, Zwas F, Antonioli DA, et al. Detection by scanning electron microscopy of a distinctive esophageal surface cell at the junction of squamous and Barrett's epithelium. Dig Dis Sci 1993;38:97-108.
4. Boch JA, Shields HM, Antonioli DA, et al. Distribution of cytokeratin markers in Barrett's specialized columnar epithelium. Gastroenterology 1997;112:760-765.
5. Chen X, Qin R, Liu B, et al. Multilayered epithelium in a rat model and human Barrett's esophagus: similar expression patterns of transcription factors and differentiation markers. BMC Gastroenterol 2008;8:1.
6. Yu WY, Slack JM, Tosh D. Conversion of columnar to stratified squamous epithelium in the developing mouse oesophagus. Dev Biol 2005;284:157-170.
7. Doupe DP, Alcolea MP, Roshan A, et al. A single progenitor population switches behavior to maintain and repair esophageal epithelium. Science 2012;337: 1091-1093.
8. Barbera M, di Pietro M, Walker E, et al. The human squamous oesophagus has widespread capacity for clonal expansion from cells at diverse stages of differentiation. Gut 2015;64:11-19.
9. Vega ME, Giroux V, Natsuizaka M, et al. Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4. Cell Cycle 2014;13:3857-3866.
10. Mills JC, Sansom OJ. Reserve stem cells: differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract. Sci Signal 2015;8:re8.
Conflicts of interest
The author discloses no conflicts.
Funding
This work was supported by US National Institutes of Health grant R01-DK097340 (D.H.W.).
© 2017 The Author. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X
http://dx.doi.org/10.1016/j.jcmgh.2017.01.015
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