A niche of trophoblast progenitor cells identified by integrin α2 is 1 present in first trimester human placentas

During pregnancy the trophoblast cells of the placenta are the only fetal cells in direct 30 contact with maternal blood and decidua. Their functions include transport of nutrients and 31 oxygen, secretion of pregnancy hormones, remodelling the uterine arteries, and 32 communicating with maternal cells. Despite the importance of trophoblast cells in placental 33 development and successful pregnancy, little is known about the identity, location and 34 differentiation of human trophoblast progenitors. We identify a proliferative trophoblast 35 niche at the base of the cytotrophoblast cell columns in first trimester placentas that is 36 characterised by integrin α2 (ITGA2) expression. Pulse-chase experiments with 5-Iodo-2ʹ- 37 deoxyuridine (IdU) imply that these cells can contribute to both villous (VCT) and extravillous 38 (EVT) lineages. These proliferating trophoblast cells can be isolated using ITGA2 as a marker 39 by flow cytometry and express genes from both VCT and EVT. Microarray expression analysis 40 shows that ITAG2 + cells display a unique transcriptional signature including NOTCH signalling 41 and a combination of epithelial and mesenchymal characteristics. ITGA2 thus marks a niche 42 allowing the study of pure populations of trophoblast progenitor cells. 43


Introduction 44
Despite the rapid growth of the human placenta in the early weeks of gestation, little is 45 known about the identity and location of a proliferative or even self-renewing niche of 46 trophoblast stem or progenitor cells. By definition, stem cells are cells capable of unlimited 47 self-renewal and differentiation into several lineages. Murine trophoblast stem cells (TSCs) 48 can self-renew in culture and contribute to all the trophoblast lineages in vivo (Tanaka et al., 49 1998). Because the existence of putative TSCs in the human placenta is unknown, we refer 50 to the proliferative cells in human placentas as trophoblast progenitors (TPs). Trophoblast 51 differentiates along two main pathways, villous and extravillous. In the first trimester, the 52 placental villus consists of a stromal core covered by two layers of trophoblast: an inner 53 Stem cells in other tissues are often characterized by expression of specific types of 59 integrins; for example, integrin β1 demarcates stem cells in epithelia and mammary glands 60 (Jensen et al., 1999;Jones and Watt, 1993;Shackleton et al., 2006;Stingl et al., 2006;Taddei 61 These findings confirm that ITGA2 can be used as a marker to isolate a unique sub-141 population of trophoblast cells from the proliferative niche at the base of the CCC. 142

Gene expression profile of ITGA2 + trophoblast 143
To understand how cells located in the proximal CCC differ from those in the villus and distal 144 CCC, the transcriptomes of the A-E-G populations from 4 placentas (G.A.= 8-9 weeks) were 145 compared by microarray. The samples cluster according to cell type by principal component 146 analysis (PCA), suggesting that there is high purity of the three populations with little 147 differences between individual donors (Fig. 4A). Hierarchical clustering provides further 148 support that the ITGA2 + cells are a distinct population from EVT and VCT (Fig. S2A). Genes 149 differentially expressed between VCT and EVT are similar to our previous findings (Fig. 150 S2B) (Apps et al., 2011). Levels for ITGA2, EGFR and HLA-G are also the highest in the ITGA2 + 151 cells, VCT and EVT respectively, further verifying the identity and purity of the isolated cell 152 types (Fig. 4B). The microarray results were validated on several candidate genes by RT-qPCR 153 (Fig. 4C). 154 As the RT-qPCR results showed that a 1.23-fold difference in gene expression on the 155 microarray was significant, we used a false discovery rate (FDR) of less than 0.05 and 1.23-156 fold difference in gene expression and found that 102 genes are more highly expressed in 157 ITGA2 + cells than the other two trophoblast populations (Table S1, Fig. 4D). Using these 158 genes for gene ontology analysis identified the terms wound healing, tissue regeneration 159 and proliferation amongst the categories with strongest significance and enrichment (Fig. 160 4E). There are far fewer genes downregulated in ITGA2 + cells ( Fig S2C, Table S2). 161 We confirmed expression of three highly expressed surface proteins in ITGA2 + cells: EpCAM, 162 TM4SF1, CD81 and COL17A1. EpCAM (also known as TACSTD1) is expressed by other stem 163 cells and more importantly, is present specifically on a subset of trophoblast progenitors in 164 the mouse placenta (Ueno et al., 2013). In human placentas, EpCAM is present on VCT and 165 at the base of the columns at 6-7 weeks, but is restricted to the base of the CCC after 8 166 weeks (Fig. 5A, n = 3, G.A.= 6-7 weeks; n = 3, G.A.= 8-10 weeks). The expression for TM4SF1 167 and CD81 is more widespread than EpCAM, but the strongest expression for both surface 168 proteins is at the base of the CCC (Fig. S3A, n = 3, G.A.= 8-9 weeks). We also identified 169 components of hemidesmosomes, COL17A1 and LAMB3 enriched in ITGA2 + cells (Fig. S3B). 170 COL17A1 is expressed mainly at the base of the CCCs and in some EVT (Fig. S3C). The 171 expression of different surface proteins in the proximal CCC will allow further analysis of the 172 heterogeneity within this niche in the future.
Apart from these surface markers, one of the top differentially expressed genes is thymosin 174 beta 4 (TMSB4X), which is associated with the stemness and differentiation of progenitor 175 and cancer cells (highlighted in Fig. 4D) (Table S1) (Bock-Marquette et al., 2009;Lv et al., 176 2013;Wirsching et al., 2013). TMSB4X increases NOTCH1 activity, and NOTCH1 is also 177 upregulated in ITGA2 + cells (highlighted in Fig. 4D) (Table S1) (Huang et al., 2016;Lv et al., 178 2013). We therefore investigated the levels of genes involved in NOTCH signalling in the 179 ITGA2 + population. NOTCH1 and downstream targets CDKN1A and PLAU are significantly 180 upregulated in ITGA2 + cells compared to VCT and EVT, suggesting that NOTCH signalling is 181 active in TPs (Fig. 5B)(Anova two-way group analysis, Tukey's multiple comparisons test). 182 HES2 expression as another target of NOTCH signalling is also enriched in ITGA2 + cells with 183 an FDR <0.05. The location of NOTCH1 in human placentas has been controversial (De Falco 184 et al., 2007;Haider et al., 2014;Herr et al., 2011;Hunkapiller et al., 2011). Our results agree 185 with Haider et al. that NOTCH1 is present at the base of the columns and demarcates a 186 progenitor-like population. 187 Since NOTCH1 is involved in the regulation of epithelial-mesenchymal transition (EMT), and 188 this pathway also came out of our gene set enrichment analyses (Fig. 5C), we looked at the 189 expression of EMT genes amongst the A-E-G populations (Fig. 5D, S3D) (Wang et al., 2011). 190 EMT genes that are 1.23 times higher in at least one of the populations were selected. Most 191 of the gene expression changes between the EGFR + and HLA-G + populations reflect the 192 epithelial and mesenchymal nature of VCT and EVT respectively, as previously described (Fig. 193 S3D) (Davies et al., 2016). The ITGA2 + subgroup of cells, however, shows an intermediate 194 phenotype between these two extremes. As such, it is more akin to VCT in terms of CDH1  expression, but at the same time expresses high levels of TPM1 and TPM2, more 196 similar to EVT cells (Fig. S3D). Moreover, we identified a particular group of  genes that are exclusively upregulated in the ITGA2 + population (Fig. 5D). 198 Taken together, the transcriptome of the A-E-G populations shows that there is a 199 proliferative population of cells at the base of the CCCs marked by ITGA2. To understand 200 more about the differentiation capacity of these cells, we performed lineage tracing on 201 human first trimester placentas. 202

Cells in the proximal column can contribute to VCT and EVT 203
Since lineage tracing by genetic manipulation is not practicable in human placentas, we used 204 IdU labelling of explants as a proxy hereditary label to trace the fate of cells. Specifically, 205 cells were treated with IdU for an hour on Day 0, and half of the explants were fixed 206 immediately while the other half were fixed after three days (Fig. 6A). With IdU portioned 207 equally between daughter progeny following division, the positional fate of cells could be 208 traced over time. 209 Following short term IdU incorporation, IdU + cells were visible in a region close to the base 210 of the CCCs as described above, localizing high proliferative activity to cells in this niche (Fig.  211 1B). To trace the fate of these proliferative cells, we then examined the pattern of IdU 212 staining after 3 days of chase. This allowed the migration of IdU labelled cells into the 213 columns to be assessed in two ways: The first method relied on the tendency of IdU + cells to 214 form "strings" of consecutive positively labelled cells in the proximal CCC. We reasoned that 215 the change in the length of the string could provide an estimate of the cell migration rate 216 from the base region. To control for potential clone variation associated with lateral division, 217 only cells in the longest string in each column were scored. In this way, we obtained an 218 estimate for the average length of the longest string per column for each donor placenta 219 between Day 0 and Day 3. An example of how cells in the string were counted is shown in 220 columns.

Discussion 235
The identity and location of TP cells in early human placentas are still unknown. We have 236 identified ITGA2 as a surface marker on a subpopulation of proliferative trophoblast residing 237 at the base of the CCC that can probably contribute to both VCT and EVT. Using it, we have 238 been able to isolate and characterise putative TPs. We used placentas from the first 239 trimester to study TPs, as expression of proliferative markers decreases with gestational age 240 in the human placenta (Arnholdt et al., 1991;Hemberger et al., 2010;Horii et al., 2016). 241 Similarly, EpCAM is expressed in VCT early in the first trimester but becomes restricted to 242 the proximal CCC at approximately 8 weeks. This evidence indicates that the proliferative 243 niche becomes more restricted as gestation proceeds in keeping with the exponential 244 growth of the placenta very early in gestation. This is analogous to the gut where 245 proliferation is initially present throughout the epithelium but becomes confined to the 246 crypts during development (Noah et al., 2011). 247 Sites of proliferation are commonly associated with progenitor cells. To locate these in early 248 pregnancy, we used both Ki67 and IdU on sections of first trimester placentas. In agreement 249 with others, we found that proliferative markers are concentrated at the base of the CCCs, 250 with less proliferation occurring in the VCT population scattered around the villi between 7-251 11 weeks (Arnholdt et al., 1991;Bulmer et al., 1988;Hemberger et al., 2010;Korgun et al., 252 2006;Lash et al., 2006;Muhlhauser et al., 1993;Westerman et al., 2004). Our lineage 253 tracing results suggest that these cells localised at the base of the CCC can contribute to 254 both VCT and EVT. Next, because the stem/progenitor cell niche is often characterized by 255 the expression of particular integrin subtypes in a range of tissues, we looked for an integrin 256 whose expression was restricted to the base of the CCC (Chen et al., 2013). We found that 257 ITGA2 marks a small group of cells in this proliferative niche and can be used to isolate the 258 putative TPs. Villous endothelial cells also express ITGA2 but these can be excluded using the 259 specific endothelial marker, CD34. ITGA2 can only form heterodimers with ITGB1, and a2b1 260 integrin binds to collagen I, II, IV, XI and XXIII as well as laminin (Tuckwell et al., 1995;261 Tuckwell et al., 1996;Tulla et al., 2001;Veit et al., 2011). Although collagen I was 262 upregulated in ITGA2 + trophoblast in our microarray analysis, we found significant 263 expression only in the villous stromal core and not in the CCC (unpublished results), thus 264 making a functional interaction with one of the other binding partners more likely. Mice 265 lacking the Itga2 gene are viable and exhibit no placental defects, suggesting that it is not 266 essential for murine trophoblast development (Chen et al., 2002). Whilst the role of ITGA2 in trophoblast cells is unclear, it also marks proliferating cells in other organs (Beaulieu, 1992;268 Lussier et al., 2000;Zutter and Santoro, 1990). 269 We used flow cytometric cell sorting to isolate the ITGA2 + cells at the base of the CCCs and 270 performed a transcriptome analysis to identify their specific expression profile. Amongst the 271 upregulated genes in the ITGA2 + trophoblast, we identified COL17A1 and LAMB3, 272 components of hemidesmosomes. COL17A1 is critical for the maintenance of hair follicle 273 stem cells and deficiency of COL17A1 leads to loss of the stem cell signature and premature 274 hair ageing (Tanimura et al., 2011). Moreover, the product of SFN (Stratifin or 14-3-3s), 275 another ITGA2 + upregulated gene, can bind both COL17A1 and keratins, therefore bridging 276 the hemidesmosomes to the rest of the cytoskeleton (Li et al., 2007). Indeed, the balance 277 between proliferation and differentiation in skin has parallels with the CCC and 278 differentiation to EVT. 279 We also find that ITGA2 + trophoblast are enriched in genes involved in the NOTCH1 280 signalling pathway, HES2, CDKN1A, PLAU and MYC. Moreover, TMSB4X as an ITGA2 + -281 enriched factor is known to increase the expression of NOTCH1, providing evidence for the 282 self-reinforcing nature of the ITGA2 niche (Huang et al., 2016;Lv et al., 2013). As expression 283 of different NOTCH receptors is restricted to subsets of trophoblast, and the inhibition of 284 NOTCH signalling elicits two opposing effects of proliferation and differentiation in a mixed 285 population of trophoblast, it will be interesting to study the specific inhibition of NOTCH1 in 286 ITGA2 + trophoblast in vitro (Haider et al., 2014). 287 As cells in the CCC move away from the basement membrane and invade the decidua upon 288 attachment to the uterus, they undergo a process typical of EMT (Davies et al., 2016). We 289 find that ITGA2 + trophoblast cells at the base of the CCC exhibit an unusual expression 290 repertoire of both epithelial and mesenchymal markers. Even within the group of genes 291 generically classified as "mesenchymal", most of them are not expressed in EVT. These data 292 suggest that the ITGA2 + subpopulation is in a unique state between epithelial-like VCT and 293 the more mesenchymal-like EVT. This phenotype is in line with the position of ITGA2 + cells at 294 the interface between both groups, supporting the notion that they may be capable of 295 contributing to both of these major trophoblast populations. 296 The flow cytometry data further corroborate this point as the ITGA2 + population is 297 proliferative and contains both EGFR + VCT and HLA-G + EVT cells. The truly bipotential 298 capacity of these cells is difficult to prove however, as neither lineage tracing of human 299 trophoblast or live imaging are possible because of the optical density of the CCCs and the 300 floating nature of the villi. We used a thymidine analogue pulse-chase strategy as a 301 preliminary attempt to study the differentiation potential of the cells in this proliferative 302 niche. Based on the contribution of IdU + cells, we conclude that the cells at the base of the 303 CCCs do contribute to both VCT and EVT, although we cannot strictly rule out the presence 304 of separate progenitor cells with different lineage contributions. 305 Taken together, we have identified a cell surface marker, ITGA2, which marks a proliferative 306 TP compartment in the first trimester placenta that is regulated by NOTCH signalling and 307 exhibits unique expression characteristics. These insights will help elucidate the putative 308 stem or progenitor cell niche in the early human placenta for future attempts at culturing a 309 self-renewing human trophoblast cell population. 310

Materials and methods 312
Ethics 313 Ethical approval was obtained from the Cambridgeshire 2 Research Ethics Committee 314 (reference no. 04/Q0108/23; Cambridge, United Kingdom) and informed written consent 315 was obtained from each patient.

Isolation of cells from human placentas 333
To obtain single cells from the placenta, the chorionic villi were scraped off from the 334 membranes and incubated in 0.2% trypsin at 37°C for 9 min. The resulting mixture was 335 sieved through muslin cloth and the flow through was centrifuged. The pellet was re- The data was analysed using FlowJo (Tree Star Inc.). 354

Extraction of RNA 358
Cells were lysed in TRIzol™ Reagent (Invitrogen #15596026) and 200 µL of chloroform was 359 added for every 1 mL of TRIzol™. The mixture was then vortexed and centrifuged at 12000 360 RCF, 4°C, for 15 min. The upper aqueous phase was transferred to a new tube, 1.5 times 361 volume of ethanol was added and the entire content was then centrifuged through 362 Purelink™ columns (Invitrogen #12183-016). RNA was purified from the columns following 363 the manufacturer's protocol, including the DNase step. The final product was eluted in 12 µL 364 of RNase-free water. 365

RT-qPCR 366
RNA was converted into cDNA using the Transcriptor First Strand cDNA Synthesis Kit from 367 Roche (#04379012001). 10 µL of 2x Fast SYBR® Green Mastermix (Applied Bioscience 368 #4385612) and 10 ng of cDNA were used per test, and each gene were tested in triplicates 369 using the Applied Biosystems® 7500 Real-Time PCR System. The settings of the machine 370 were: 95°C 5 min, then 40x (95°C 10 s, 60°C 30 s). Melting curve analysis was done at the 371 end, to ensure that there was only one amplicon. 372

Microarray 376
Four donor sets of three cell types with RNA integrity number (RIN) above 7 as measured by 377 2100 Bioanalyzer Instrument (Agilent Technologies) were amplified using the Ovation® Pico 378 WTA System v2 kit (NuGEN, #3302) according to the manufacturer's protocol. Good quality 379 final products as assessed by the 2100 Bioanalyzer Instrument were biotinylated using the 380 Encore Biotin Module by NuGEN (#4200-12) and hybridised to Illumina Human HT-12 V4 381 BeadArray (#BD-103-0204) using the manufacturer's protocol. After correcting for 382 background, the output from Illumina BeadStudio were logged to the base of two and 383 multiple testing corrections was applied to paired-wise analysis of each cell type against 384 each other in the R statistical programme. The data was filtered to remove probes for which 385 the detection p-value was not above 0.01 for at least one sample for the analysis.  We thank all donors for providing tissue and Diane Moore for coordinating the tissue 418 collection. We are also grateful to Nigel Miller and Julien Bauer for their expertise on flow 419 cytometric sorting and analysis of microarray data respectively. 420

Competing interests 421
No competing of interest declared.