261WEI ET AL. Biol Res 41, 2008, 261-270Biol Res 41: 261-270, 2008 BRHematopoietic potential of mouse placenta with theapplication of placenta flushing

BIN WEI1, SUOMAO YUAN2, YINGMAO GAO1,* and LUJUN BING1

1 Histology and Embryology Institute, Medical School of Shandong University, Jinan, Shandong, 250012, PRChina2 Peking Union Medical College Hospital, Chinese Academy of Medical Science & Peking Union MedicalCollege, Beijing, 100730, PR China

ABSTRACT

Several major vascular tissues, such as the aorta-gonad-mesonephros region (AGM), yolk sac, and fetal liverhave been confirmed to possess hematopoietic function. Recently, the placenta has been demonstrated asanother hematopoietic organ. However, it is not conclusive whether the placenta possesses hematopoieticability. Therefore, we undertook a series of experiments to study the hematopoietic functions of placenta.Fetal blood circulation in the placenta is difficult to be eliminated and its interference in the study of placentalhematopoiesis is inevitable. With the application of placental flushing, fetal blood contained in the placentawas eliminated. We then made the further study of placental hematopoiesis after the E12.5 placenta wasflushed. Our studies showed that placental cells expressing Sca-1, CD117 and CD34 were mainly restricted tothe embryonic vessels of E12.5 placenta. The results of fluorescence activated cell sorter (FACs) analysis andcolony forming cells (CFC) assay demonstrated that both placenta and placental blood containedhematopoietic stem/progenitor cells (HS/PCs), including CFU-GMs, CFU-GEMMs, BFU-Es, and HPP-CFCs.The frequency of HS/PCs in the placenta was 2-3 times that of placental blood. Therefore, it is necessary toclear placental blood out of the placenta in the studies of the hematopoietic potential of placenta. The placentastill possessed the hematopoietic potential after the fetal blood is flushed out. These observations providefurther evidences that the placenta is a hematopoietic organ, as has been proposed for other embryonichematopoietic sites.

Key terms: placenta, hematopoiesis, HS/PCs, mouse

* Corresponding author: Yingmao Gao, Wenhua West Road 44#, Jinan, Shandong, PR China. Telephone +86 (0)53188380491. E-mail address:yingmaogao@hotmail.com.

Received: March 17, 2008. In Revised Form: July 3, 2008 Accepted: October 17, 2008

INTRODUCTION

The ontogeny of the hematopoietic systemin vertebrates is characterized by sequentialactivity of distinct hematopoietic organs.Coordinated activities of the differenthematopoietic sites ensure both rapidproduction of differentiated blood cells forthe embryo’s immediate needs and theestablishment of a large pool ofundifferentiated hematopoietic stem cells(HSCs). Yolk sac, fetal liver, thymus,spleen and bone marrow have beenidentified as the organs carrying out thisfunction in mammalian embryos.

Recently, a series of studies havesuggested that the fetal placenta is also a

hematopoiet ic organ that plays animportant role in establishing the HSCpool during development. The study byAlvarez-Silva and colleagues shows thatthe placenta contains a high frequency ofmulti-potential colonogenic progenitors,before l iver colonizat ion ini t iates(Alvarez-Silva et al., 2003). Furthermore,subsequent in vivo studies show thatmidgestation placenta also harbors a largepool of pluripotent HSCs that have thecapacity to self-renew and repopulate theentire hematopoietic system in irradiatedadult hosts (Gekas et al., 2005; Ottersbachand Dzierzak, 2005). These studies haveestablished the placenta as yet anotherhematopoiet ic organ that plays an

WEI ET AL. Biol Res 41, 2008, 261-270262

important role in HSC development.However, the aforementioned studies donot give clues to whether the placenta issimply a colonization site (like the fetalliver), or whether it is also a source ofhematopoietic cells. The studies of Zeigleret al (2006) and Corbin et al (2007)demonstrated that the two precursors ofthe placenta, the allantois and the chorion,possessed hematopoietic potential beforetheir fusion into placenta. Thus, it seemsthe placenta maintains its hematopoieticpotential from i ts formation to themidgestation. However, previous studieshave shown that the vasculature system ofthe placenta is established several daysafter the formation of placenta, and thatumbil ical blood contained a highpercentage of HS/PCs. These facts make itunclear whether the hematopoiet icpotential of the midgestation placenta wasfrom placental cells, or from fetal blood,or from both. Consequently, we developedthe technique of placenta flushing to clearfetal blood cells in the placenta beforemaking further s tudy of placentalhematopoiesis.

MATERIALS AND METHODS

Mice

C57BL/6 mice were mated overnight, andthe day of vaginal plug was considered asday 0.5 of embryonic development (E0.5).The animals were housed according toinstitutional guidelines, with free access tofood and water. Animal procedures werecarried out in compliance with theStandards for Humane Care and Use ofLaboratory Animals.

Placental flushing

Pregnant females of E12.5 were killed bycervical dislocation. The uterine wasremoved from the conceptuses, washedextensively in D-Hank’s balanced saltsolution. The uterine was opened andembryos at E12.5 were harvested. Theentirety of embryo, umbilical cord andplacenta was maintained intact. Placenta

flushing was performed immediately afterthe embryos were removed from theuterine. Under a stereomicroscope(Olympos, JPN), the umbilical artery andvein were identified by the direction ofblood flow. A fine needle (BD29 G) wasinserted into the umbilical artery with thetip pointing to the placenta. Heparin saline(concentration 0.5 mg/ml, 290000 IU/ml)was used as flushing fluid and injected intothe umbilical artery slowly (0.1ml/min) toflush the embryonic vessel system of theplacenta. The flushed blood from theplacenta was collected for the further assay.To quantify and determine the flushingeffect, thirty placentas were collected anddivided into six groups at random. In thesix groups, the volumes of flushing fluidused were 0 ml, 0.05ml, 0.10 ml, 0.15 ml,0.20 ml, and 0.25 ml, respectively. Then theplacentas were serially cryosectionedcoronally at 10μm. One slice in every tenserial slices was collected and total 20slices for each placenta were harvested toevaluate the flushing effect. Afterhematoxylin-eosin staining, ten visualfields (×400) were selected randomly foreach slice. Then the fetal blood cells in theembryonic vessels were counted in eachvisual field and the data were recorded forstatistical analysis with the one-wayANOVA, using the SPSS software package(SPSS, USA).

Placentas dissection, immunofluorescence,and confocal microscopy

Placentas of E12.5 were dissected frommaternal tissue. Frozen sections (10 μm)were collected on plus slides and were fixedby acetone. Slides were then stored at 4 oC,until ready for use. The sections wereincubated with rat monoclonal anti-mouseCD34 (Serotec, UK), CD117 (Biolegend,USA), and Sca-1 (Biolegend, USA) for 1hour at 37 oC. The sections were incubatedfor a further hour at room temperature influoresceinisothiocyanate (FITC)-labeledgoat anti-rat IgG (KPL, UK). A negativeisotype matched control was prepared byreplacing the CD34 primary antibody withPBS with the same concentrations as thoseof the primary antibodies. A positive

263WEI ET AL. Biol Res 41, 2008, 261-270

control section of mouse fetal liver wasincluded in each batch of staining.

Single cell suspension preparations anddensity gradient centrifugation

To functionally assess hematopoieticactivity of the flushed placenta, theplacentas (free of umbilical cord, remnantsof the yolk sac and maternal decidua) weredissected and dissociated into single cellsuspension. The placentas weremechanically dissected free and drawnthrough a 16 G needle and incubated with0.1% collagenase in 10% fetal calf serum(FCS) / phosphate-buffered saline (PBS) for30 minutes at 37°C and trypsin for 10minutes at 37°C. Then the Percoll(Amersham, UK) fluids of different density(1.10/ml, 1.080g/ml, 1.055g/ml) wereadded into the centrifuge tube one by onefrom higher density to less, and then thecell suspension was gently placed at theutmost upper layer. After centrifugation for25 minutes with 2000 rpm, the cellsresiding between the top layer and themiddle layer were harvested. The purifiedsingle cell suspension was counted andviability was tested via Trypan blueexclusion criteria. Meanwhile, the flushedblood cells from the placenta were preparedand treated according to this procedure.

Fluorescent antibody surface staining andflow cytometry

All antibody stainings were carried out ina volume of 100-200 μl PBS/10% FCS onice for 30 minutes with the followingantibodies (Biolegend, USA): anti-CD34-PE, anti-CD117-PE, and anti-Sca-1-FITC.Labeled cells were finally washed twice inPBS, and filtered through a nylon meshscreen to remove debris. The cells wereanalyzed using a f low cytometry(Beckman Coulter, USA). The data wereanalyzed using the System II version 3.0.Each value was obtained from the meansof s ix independent experiments. Todetermine background levels, cells werestained with fluorochrome-conjugatedimmunoglobulin isotype controls fromBiolegend.

Clone forming cells (CFC) and highproliferation-potential colony-forming cells(HPP-CFC) assays

For analysis of progenitor activity in theE12.5 flushed placenta, 105 purified singlecells were plated per 35 mm dish inMethoCult M3434 methylcellulose media(Stem Cell Technologies, CAN). Thecommercial medium containsmethylcellulose in IMDM [15% fetal calfserum (FCS), 1% bovine serum albumin(BSA), 10-4 M 2-mercaptoethanol, 2 mMglutamine, 3 U /ml recombinant human (rh)erythropoietin, 10 ng /ml rh interleukin 6(IL-6), 10 ng /ml recombinant mouse (rm)IL-3, 50 ng /ml rm Stem Cell Factor (SCF),10 mg /ml rh insulin and 200 mg /ml humantransferrin (iron saturated)]. After 14 daysof culture at 37°C in a humid atmospherecontaining 5% CO2, colonies wereidentified based on their morphology underan inverted microscope and scored. Eachvalue was obtained from the means of sixindependent assays.

To check for the presence of long-termHPP-CFCs, first passage colonies with adiameter exceeding 0.5 mm were pluckedafter 20-25 days of culture, pooled andwashed with PBS. Cells were counted and105 cells were replated per dish inMethoCult M3434 medium. The colonieswere replated three times according to thesame protocol and scored 60 days afterinitial seeding.

RESULTS

The effect of placenta flushing

The unflushed placenta was full of fetalcirculation blood. After flushing, fetalcirculation blood was cleared out of theplacenta (Fig 1A). Under microscope, wefound fetal blood cells that were previouslytrapped in the embryonic vessels (Fig 1B)had been completely flushed out of theplacenta, and placenta flushing caused noexpansion of the vessel lumina. Thestructure of endothelial cells of theembryonic vessels and other cells stillremained intact and the tissue space was

WEI ET AL. Biol Res 41, 2008, 261-270264

normal (Fig 1C). The relationship betweenthe flushing volume and the flushing effectis shown in Fig 1D. The results of statisticalanalysis demonstrated that the differencesbetween the first five groups(0 ml, 0.05 ml,0.10 ml, 0.15 ml, and 0.20 ml) weresignificant (P<0.05), while the differencebetween the last two groups (0.20 ml and0.25 ml) was not statistically significant(P>0.05). In our subsequent experiments,we flushed each placenta with 0.2 mlflushing fluid.

Phenotype and localization of placental HS/PCs

At E12.5, CD34 stained the endothelialcells lining the vessels in the placentalabyrinth (Fig 2A). CD117+ cells weremainly found in the endothelial cells liningthe vessels in the placenta labyrinth and themesenchymal tissue of the chorionic plateand the labyrinth (Fig 2B). Most strikingly,a high level of Sca-1 expression was foundin the embryonic vessels of the placenta(Fig 2C and D). Sca-1 expression was in theendothelial cells lining the fetal vessels thatformed a network through the labyrinthregion. It was also expressed by a fewendothelial cells lining the vessels in the

chorionic plate. At E12.5, in the endothelialcells of the umbilical artery, the expressionof CD34 and Sca-1 could also be detected(Fig 1E and F), while the expression ofCD117 could not be detected.

The number and phenotypic analysis of theisolated and purified cells in the placentaand placental blood

First, we isolated and purified the placentalcells with Percoll fluid. We then performedflow cytometric analysis to determine thenumber and phenotypic characteristics ofthe purified placental cells in the placentaand placental blood. Placental cells werestained with antibodies specific for HS/PCmarkers: Sca-1(HSC marker), CD34(endothelial and HSC marker), and CD117(HSC and immature, hematopoieticprogenitor marker). The percentages of thethree cell populations in the total purifiedplacental cells were 20.2%, 24.6%, and26.2% respectively. For purified placentalblood cells, the percentages of the threepopulations were 8.2%, 6.3%, and 6.5%,respectively. The concentration of positivecells in placental cells was higher than inplacental blood. The absolute numbers ofpositive cells of CD34, CD117 and Sca-1

Fig. 1: Unflushed and flushed placentas. (A)The left placenta was full of fetal blood beforeflushing. The right is a flushed placenta. The upper layer was the fetal part. (B) In the section ofE12.5 unflushed placenta, hematoxylin-eosin staining showed many vessels surrounded by a thinendothelial layer, which were the embryonic/fetal vessels in the placental labyrinth. The lumen ofmany vessels of the embryonic vessels in the placental labyrinth was filled with packederythroblasts. (C) In the section of the flushed placenta, there were no blood cells left in the lumenof blood vessels of the embryonic vessels in the placental labyrinth. White arrows show the lumenof the embryonic vessels in the placental labyrinth. (D) The relationship between the flushingvolume and the flushing effect. The cell number in the fetal blood vessels/visual field was theaverage of the total visual fields of the five placentas in each group. For each placenta, 200 visualfields were counted. The flushing speed was 0.1 ml/min. Chorionic plate (CP), placenta labyrinth(Lb).

265WEI ET AL. Biol Res 41, 2008, 261-270

Fig. 2: Expression of CD34, CD117, and Sca-1 in the placenta and umbilical artery. (A)Coronal section of E12.5 placenta was stained with antibodies against CD34. Arrows indicate theendothelial cells of embryonic vessels. (B) Coronal section of E12.5 placenta was stained withantibodies against CD117. Arrow shows the endothelial cells of embryonic vessels. Arrowheadindicates the mesenchymal cells of the placenta labyrinth. (C) Coronal section of E12.5 placentawas stained with antibodies against Sca-1. Arrow indicates the endothelial cells of embryonicvessels. (D) Transverse section of E12.5 placenta was stained with antibodies against Sca-1. Arrowshows the endothelial cells of embryonic vessels. (E) Longitudinal section of E12.5 umbilicalvessels stained with anti-CD34 antibody. Arrow indicates the endothelial cells of umbilical artery.(F) Longitudinal section of E12.5 umbilical vessels stained with anti-Sca-1 antibody. Arrow showsthe endothelial cells of umbilical artery. (A-F) Sections were analyzed by confocal microscopy. cp,chorionic plate; l, labyrinth; ua, umbilical artery.

were 1.4×104, 1.8×104, and 1.8×104

respectively in purified placental cells, andwere 0.7×104 0.5×104, and 0.5×104

respectively in purified placental blood(Table 1). The number of positive cells inthe purified placental cells was higher thanin purified placental blood. For the cellsubpopulation of CD34+ /Sca-1+, thepercentage in purified placental cell(15.3%) was also higher than in placentalblood (5.1%). In placental cells, 58% ofSca-1+ cells expressed CD34. In placentalblood, 78% of Sca-1+ cells expressed CD34(Fig 3).

Frequency of short-term progenitors

These experiments were carried out usingE12.5, isolated and purified cell preparations.Three different types of progenitors – CFU-GEMMs (colony forming units, granulocytes,erythrocytes, monocytes, macrophages),BFU-Es (burst-forming units, erythroid) andCFU-GMs (colony-forming units,granulocytes, macrophages) – diagnosed fromthe colony phenotype, were scored separatelyon day 14 of culture (Fig. 4). CFU-GEMMsexceeding 0.5 mm in diameter (HPP-CFCs)were selected for serial replating.

WEI ET AL. Biol Res 41, 2008, 261-270266

TABLE 1

The cell number of single cell suspension, isolated and purified cells and FACS assay influshed placenta and placental blood at E12.5.

Examined tissues Absolute number(x±s) Percentage(x±s %)

Total cell number of singlecell suspension /PL Flushed PL 6.2±0.4×107 -

PB 3.3±0.3×107 -

Number of isolated andpurified cells /PL Flushed PL 0.7±0.2×105 -

PB 0.7±0.5×105 -

FACS assay Flushed PL CD34+ 1.4×104 20.2±3.0CD117+ 1.8×104 24.6±6.2Sca-1+ 1.8×104 26.2±5.2

CD34+/Sca-1+ 1.1×104 15.3±4.1PB CD34+ 0.6×104 8.2±3.5

CD117+ 0.5×104 6.3±2.8Sca-1+ 0.5×104 6.5±3.3

CD34+/Sca-1+ 0.4×104 5.1±3.1

PL: placenta; PB: placental blood.

Fig. 3: Flow cytometric analysis of E12.5 placenta and placental blood. Isolated and purifiedcells from E12.5 placenta and placental blood were stained with phycoerythrin-conjugatedantibodies against CD34 as indicated on the y axis and FITC- conjugated antibodies against Sca-1as indicated on the x axis. Percentages for each population are indicated inside the quadrants.

267WEI ET AL. Biol Res 41, 2008, 261-270

After culturing for 14 days, the colonyyields of BFU-Es, CFU-GMs, and CFU-GEMMs in placental cells were higher thanin placental blood. The frequency ofplacental BFU-Es was at least two timeshigher than that of placental blood BFU-Es.The frequency of CFU-GEMMs in theplacenta was also two times as high as in theplacental blood. Nevertheless, the frequencyof the placental CFU-GMs was only onetime higher than that of the placental bloodCFU-GMs. The characteristics of themorphology of placental BFU-Es wasmulticentered and highly hemoglobinized,while placental blood BFU-Es displayedonly weakly hemoglobinized clusters (Fig.4A, B). In the case of CFU-GMs, placentalCFU-GMs were dense, medium to largesized, and often actually so large that theyseemed to result from the fusion of clusters,whereas placental blood CFU-GMs weresmall sized(Fig. 4C, D). Both placentalCFU-GEMMs and placental blood CFU-

GEMMs were huge, and so qualified moreadequately as HPP-CFCs. Compared toplacental blood CFU-GEMMs, placentalCFU-GEMMs appeared larger, denser, andcontained more cells, with a very large coreof hemoglobinized cells (Fig. 4E, F).

Long-term HPP-CFCs

In order to monitor the self-renewal capacityof hematopoietic progenitors in the placentaand placental blood, we replated the verylarge multilineage colonies derived fromHPP-CFCs every 2 weeks for 2-3 months. Ateach replating, some very large colonies wereagain obtained. When the colonies werescored at 60 days, 35 CFU-GEMMs can beobtained from 105 replated placental cells. Bycontrast, HPP-CFCs could also be obtainedfrom the placental blood and the frequencywas about two-thirds of the placenta (Fig 5).Furthermore, the placental blood colonieswere smaller than the placental colonies.

Fig. 4: CFU-GEMM, BFU-E, CFU-GM colonies obtained from E12.5 placenta and placentalblood after 14 days of culture in methylcellulose-based medium. (A) Placental BFU-E colonieswere multicentered and highly hemoglobinized. (B) Placental blood BFU-Es displayed only weaklyhemoglobinized clusters. (C) Placental CFU-GM colonies were dense, medium to large sized, andoften actually so large that they seemed to result from the fusion of clusters. (D) Placental bloodCFU-GMs were small-sized. (E) Placenta CFU-GEMM colonies were characterized by largediameter exceeding 0.5mm, with a very large core of hemoglobinized cells. (F) Placental bloodCFU-GEMMs were less hemoglobinized.

WEI ET AL. Biol Res 41, 2008, 261-270268

DISCUSSION

Proper function of the placenta is essentialfor the development of the growingembryo. In previous studies, the placentahas not been regarded as taking part in fetalblood formation. Recently, a series ofstudies suggest that the fetal placenta isalso a hematopoietic organ that plays animportant role in establishing the HSCspool during development. Alvarez-Silva etal (2003) examined the progenitors in E8-E17 placenta and found that the progenitorsbegan to appear at the stage of 18 sp (E8.5)in the placenta. To examine the placenta forpotent, adult-engrafting HSCs, Ottersbachand Dzierzak (2005) examined E9-E12placenta (without the decidua or umbilicalvessels). It was shown that no HSCs werefound in E9 or E10 placenta. However,potent placental HSCs began to be detectedat E11 and were present at high levels atE12.

As is known, the placenta labyrinthcontains abundant fetal circulation blood.From E10.0 the definitive hematopoieticprogenitors prefer to begin to freely

distribute within the embryonic vasculature(McGrath et al. , 2003). Long-termreconstituting HSCs appears in circulationby E12.5 (Christensen et al. , 2004).Kumaravelu et al. (2002) found that at E12there are 3.2 HSCs in circulation. In ourstudy, we performed short-term progenitorassay and FACs with the flushed placentalblood. The results showed the bloodtrapped in the E12.5 placenta contained aquantity of progenitors, which was about 1/3-1/2 of that in E12.5 placenta. Therefore,fetal blood cells trapped in the placentashould be one of the important factors to beconsidered in the study of placentalhematopoiesis. However, how to clear fetalcirculation blood in the placenta and detectthe function of placental HS/PCs is still aproblem. Consequently, we developed thetechnique of placenta flushing. In ourresult, there was no significant difference influshing effect between 0.2 ml group and0.25 ml group. It seemed that more flushingfluid than 0.20ml could not bring more fetalblood cells out. But remnant fetal bloodcells in the placenta were not more than2%, and the fetal blood could be nearly

Fig. 5: Short-term progenitors assay and long-term HPP-CFCs assay. 105 isolated and purifiedcells were used for short-term progenitors assay in both placenta and placental blood. After 14days of culture, colonies were identified based on their morphology and scored. HPP-CFCs weretaken from MethoCult M3434 cultures every 20-25days. 105 cells from pooled colonies werereplated at each passage. HPP-CFCs were scored after a total 60 days in culture. Each result is themean±s.d. from six independent experiments.

269WEI ET AL. Biol Res 41, 2008, 261-270

cleared out of the placenta with 0.2 mlflushing fluid.

First, we observed the distribution ofHS/PCs in the mouse placenta using theHSC surface markers. Byimmunohistochemical analysis, it wasshown that high-level Sca-1 expression wasrestricted to the embryonic vessels of theplacental labyrinth. Moreover, other HSCmarkers also were colocalized in theembryonic vessels of the placental labyrinthregion. Furthermore, we performedimmunohistochemical analysis of umbilicalcord with the same HSC surface markers. Itwas found that the HS/PCs were alsorestricted to the endothelium of theumbilical artery.

To examine hematopoietic activity, it isessential to first isolate placental cells withhematopoietic function. Continuousenzymatic digestion with collagenase andpancreatin was used to prepare placentalcell suspension. To obtain highlyconcentrated placental HS/PCs, the singlecell suspension was isolated and purifiedwith Percoll fluid density gradientseparation. After purification, theconcentration of Sca-1+ cells could beraised to 26.2% at E12.5, while Sca-1+ cellsrepresented only 3.09% of the unpurifiedtotal cell population at E12 in Ottersbachand Dzierzaks’ report (Ottersbach andDzierzak, 2005). Therefore, Percoll densitygradient centrifugation method could beused to isolate and purify placental HS/PCs.The further analysis of the hematopoieticpotential of the purified placental cellsshowed that the placenta of midgestationcontained clonogenic hematopoieticprogenitors, including CFU-GMs, CFU-GEMMs, BFU-Es, and HPP-CFCs.

The murine allantois contributes thefetal vascular and associated stromalcomponents of the placenta, including theumbilical vessels (Downs et al., 1998). AtE8.5, the murine allantois makes contactwith the chorion (Cross, 1998; Cross et al.,2003a, 2003b; Han and Carter, 2001;Rossant and Cross, 2001). The allantois hasbeen identified as a third site ofhematopoiesis in the chick embryo(Caprioli et al., 1998). However, in murineembryos, it is not yet clear whether the

allantois is hematopoietic (Downs andHarmann, 1997). Brandon M. Zeigler et al.(2006) investigated the hematopoieticpotential of the two individual componentsof the murine placenta, the allantois and thechorion, prior to establishment of vascularcontinuity within the conceptus. Theirstudies demonstrated that the allantois andchorion, isolated prior to the establishmentof circulation, have the potential to giverise to myeloid and definitive erythroidcells following explant culture. CatherineCorbel et al. (2007) examined thehematopoietic potential of the prefusionallantois and provided evidence that theallantois has hematopoietic potential per se.Since the embryonic vessels of the placentawere derived from the allantois, and thehematopoietic potential of midgestationplacenta was mainly restricted to theembryonic vessels of the placentallabyrinth, it may be reasonable to infer thatthe hematopoietic potential of themidgesation placenta was the continuity ofthe hematopoietic potential of allantois.

In conclusion, both placenta andplacental blood contain a quantity of HS/PCs in the midgestation placenta. It isnecessary to clear the placental blood out ofthe placenta in studies of the hematopoieticpotential of the placenta. The midgestationplacenta still possesses hematopoietiapotential after the fetal blood is flushed out.

ACKNOWLEDGMENTS

We thank the members of the laboratory forassistance in various parts of these studiesand for stimulating discussions, and FaliangZhu for assistance with multicolor flowcytometry. We especially acknowledge theexcellent assistance with microscopyprovided by Dr. Yuji Guo. All other authorsremain free from any conflicts of interest.

REFERENCES

ÁLVAREZ-SILVA M, BELO-DIABANGOUAYA P,SALAÜN J, DIETERLEN-LIÈVRE F (2003) Mouseplacenta is a major hematopoietic organ. Development,130: 5437-5444

CAPRIOLI A, JAFFREDO T, GAUTIER R, DUBOURG C,

WEI ET AL. Biol Res 41, 2008, 261-270270

DIETERLEN-LIEVRE F (1998) Blood-borne seedingby hematopoietic and endothelial precursors from theallantois. Proc Natl Acad Sci U S A, 95: 1641-1646

CHRISTENSEN JL, WRIGHT DE, WAGERS AJ,WEISSMAN IL (2004) Circulation and chemotaxis offetal hematopoietic stem cells. PLoS Biol, 2: E75

CORBEL C, SALAUN J, BELO-DIABANGOUAYA P,DIETERLEN-LIEVRE F (2007) Hematopoieticpotential of the pre-fusion allantois. Dev Biol, 301:478-488

CROSS JC (1998) Formation of the placenta andextraembryonic membranes. Ann N Y Acad Sci,857:23-32

CROSS JC, BACZYK D, DOBRIC N, HEMBERGER M,HUGHES M, SIMMONS DG, YAMAMOTO H,KINGDOM JC (2003) Genes, development andevolution of the placenta. Placenta, 24:123-130

CROSS JC, SIMMONS DG, WATSON ED (2003)Chorioallantoic morphogenesis and formation of theplacental villous tree. Ann.N Y Acad. Sci, 995: 84-93

DOWNS KM, HARMANN C (1997) Developmentalpotency of the murine allantois. Development, 124:2769-2780

DOWNS KM, GIFFORD S, BLAHNIK M, GARDNER RL(1998) Vascularization in the murine allantois occursby vasculogenesis with out accompanyingerythropoiesis. Development, 125: 4507-4520

GEKAS C, DIETERLEN-LIEVRE F, ORKIN SH,

MIKKOLA HK (2005) The placenta is a niche forhematopoietic stem cells. Dev Cell, 8:365-375

HAN VK, CARTER AM (2001) Control of growth anddevelopment of the feto-placental unit. Curr OpinPharmacol, 1: 632-640

KUMARAVELU P, HOOK L, MORRISON AM, URE J,ZHAO S, ZUYEV S, ANSELL J, MEDVINSKY,A(2002) Quanti tat ive developmental anatomy ofdefinit ive haematopoietic stem cells/ long-termrepopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac incolonisation of the mouse embryonic l iver.Development, 129:4891-4899

MCGRATH KE, KONISKI AD, MALIK J, PALIS J (2003)Circulation is established in a stepwise pattern in themammalian embryo. Blood, 101: 1669-1676

OTTERSBACH K, DZIERZAK E (2005) The murineplacenta contains hematopoietic stem cells within thevascular labyrinth region. Dev Cell, 8: 377-387

ROSSANT J, CROSS JC (2001) Placental development:lessons from mouse mutants. Nat Rev Genet, 2:538-548

ZEIGLER BM, SUGIYAMA D, CHEN M, GUO Y,DOWNS KM, SPECK NA (2006) The allantois andchorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential .Development, 133: 4183-4192