Experimental Hematology 35 (2007) 146–154

Simultaneous generation of CD34þ primitivehematopoietic cells and CD73þ mesenchymal stem cells from

human embryonic stem cells cocultured with murine OP9 stromal cells

Parul Trivedia and Peiman Hemattia,b

aDepartment of Medicine, University of Wisconsin-Madison, School of Medicine and Public Health,

Madison, Wis., USA; bUniversity of Wisconsin Paul P. Carbone Comprehensive Cancer Center, Madison, Wis., USA

(Received 16 June 2006; revised 6 September 2006; accepted 7 September 2006)

Objective. Human embryonic stem cells (hESCs) have been shown to generate CD34+ primi-tive hematopoietic cells after several days of coculturing with the OP9 murine stromal cellline. CD73+ multipotent mesenchymal cells have also been isolated from hESC/OP9 coculturesafter several weeks. We hypothesized that generation of CD34+ hematopoietic cells and CD73+

mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generationof CD73+ cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34+ cells aregenerated.

Materials and Methods. We cocultured hESCs with OP9 cells and examined the time course ofappearance of human CD34+ and CD73+ cells using flow cytometry. We tested the hematopoi-etic progenitor potentials of CD34+ cells generated using hematopoietic colony-formingassays, and the multipotent mesenchymal properties of CD73+ cells generated using in vitrodifferentiation assays.

Results. We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34+ he-matopoietic and CD73+ MSC generation follows a similar pattern. We sorted the CD34+ cellsand showed that they can generate hematopoietic progenitor colonies. Starting with cocul-tured cells on day 8, and through an enrichment procedure, we also could generate a purepopulation of MSCs. These hESC-derived MSCs had typical morphological and cell surfacemarker characteristics of adult bone marrow-derived MSCs, and could be differentiated to-ward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs.

Conclusions. OP9 cells when cocultured with hESCs support simultaneous generation ofCD34+ primitive hematopoietic cells and CD73+ MSCs from hESCs. � 2007 InternationalSociety for Experimental Hematology. Published by Elsevier Inc.

Since the first derivation of human embryonic stem cells(hESCs) by James Thomson in 1998 [1], there has been ex-ponential interest in the potential use of these cells in regen-erative medicine. However, before any human therapeuticapplications can be achieved, there must be reproducible,efficient, and safe methodologies for directed differentia-tion of hESCs into desired cell types, either in vitro or invivo. The derivation of primitive or differentiated bloodcells, originally from murine ESCs and, more recently,from hESCs, has been the subject of intensive research,

Offprint requests to: Peiman Hematti, M.D., University of Wisconsin

Paul P. Carbone Comprehensive Cancer Center, Hematology Office H4/

534 CSC-5156, 600 Highland Avenue, Madison, WI 53792-5156;

E-mail: pxh@medicine.wisc.edu

0301-472X/06 $–see front matter. Copyright � 2007 International Society for

doi: 10.1016/j.exphem.2006.09.003

with the ultimate goal of developing different types ofblood cells from this novel source for transplantation/trans-fusion purposes [2–4]. Methodologies used to derive hema-topoietic cells from ESCs include embryoid body formation[5–7]; coculture with a variety of hematopoietic supportivecells, such as stromal cells of bone marrow origin [8–10];or a combination of both [11]. More than a decade ago, mu-rine bone marrow-derived OP9 stromal cell line was shownto be supportive of the generation of primitive hematopoi-etic cells from murine ESCs [12] and since then this cellline has been extensively used in murine ESC-derived he-matopoietic studies [13,14]. This stromal cell line derivedfrom newborn op/op mouse calvaria does not produce func-tional macrophage colony-stimulating factor (M-CSF) be-cause of an osteopetrotic mutation in the gene encoding

Experimental Hematology. Published by Elsevier Inc.

147P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

M-CSF. More recently, the OP9 cell line has also been usedfor efficient derivation of primitive hematopoietic cellsfrom hESCs [10,15], as well as from nonhuman primateESCs [16,17]. In this experimental system, CD34þ cellsare usually generated from ESCs after several days of co-culturing with OP9 cells, and then decline by 2 weeks. In-terestingly, Barberi et al. [18] recently reported that 40 daysafter coculturing hESCs with OP9 cells an average of 5% ofcocultured cells were CD73þ cells of human origin [18].These cells were sorted, expanded, and shown to be multi-potent mesenchymal precursors by their capability to differ-entiate into multiple mesenchymal derivatives, such asosteogenic, adipogenic, and chondrogenic cells.

Because of the close ontogenic relationship between he-matopoietic and their supportive mesenchymal stem (orstromal) cells (MSCs) [19,20], we hypothesized that thesetwo types of cells might follow a similar pattern of gener-ation in the hESC/OP9 coculture system. In this study, weinvestigated and characterized the kinetics of generationof primitive hematopoietic cells and MSCs from hESCscocultured with OP9 cells.

Material and methods

hESC and OP9 cell culturesThe hESC cell lines H1 and H9 (federally registered as WA01 andWA09), passages 25–35 and karyotypically normal were obtainedfrom WiCell (Madison, WI, USA), and maintained in an undiffer-entiated state by passaging on matrigel plates in mouse embryonicfibroblast (MEF)-conditioned media. New hESC lines expressinggreen fluorescent protein (GFP) were derived using the plasmidand methodology described by Liu et al. [21]. The OP9 cell linewas purchased from American Type Culture Collection (Mana-ssas, VA, USA) and was maintained on gelatinized plates in a-modified minimum essential (a-MEM) medium (Invitrogen,Carlsbad, CA, USA) containing 20% fetal bovine serum (FBS;Hyclone Laboratories, Logan, UT, USA), 0.1 mM nonessentialamino acid (NEAA), and 2 mM L-glutamine (glu).

Coculture differentiation towardhematopoietic and mesenchymal cellsDifferentiation of hESCs toward hematopoietic and mesenchymalcells was induced by plating hESCs at a density of 1 � 105 cellsmL onto six-well plates containing a confluent monolayer of OP9cells irradiated with 80 Gy, and differentiation medium containinga-MEM/10% FBS/NEAA/glu and 0.1 mM b-mercaptoethanol.Cocultured cells were then incubated at 37�C/5% CO2 with halfmedium change on days 4, 6, and 8.

Flow cytometryFor flow cytometry, cocultured hESC/OP9 cells were collected atdifferent time points after treatment with collagenase-IV (Invitro-gen) followed by 0.05% trypsin/0.5 mM ethylenediamine tetraace-tic acid (EDTA); similarly, mesenchymal cells generated laterwere collected after treatment with trypsin/EDTA. Dissociatedcells were centrifuged and washed with phosphate-buffered saline(PBS) supplemented with 2% FBS and 0.1% sodium azide, and

single-cell suspensions were prepared. Only human specificmonoclonal antibodies tested to be nonreactive to OP9 cellswere used for labeling of single-cell suspensions, includingCD73 (IgG1, phycoerythrin [PE]), CD29 (IgG1, PE), CD34(IgG1, PE), CD44 (IgG2b, PE), CD45 (IgG1, PE), CD54 (IgG1,PE), CD90 (IgG1, allophycocyanin) and CD105 (IgG1, allophyco-cyanin) (all from BD Biosciences (San Jose, CA, USA). Controlstaining, with appropriate isotype-matched monoclonal antibodiesalong with unstained control samples, was included in each exper-iment. Samples were analyzed using a FACSCalibur flow cytom-eter with Cell Quest acquisition software (BD Biosciences). Listmode files were analyzed by FlowJo software (Tree Star, Ashland,OR, USA).

CD34þ cell sorting andhematopoietic colony-forming cell assaysSingle-cell suspensions from day 8 of cocultures of hESCs withOP9 cells were labeled with CD34 paramagnetic monoclonal an-tibodies using Direct CD34 Progenitor Cell Isolation kit (MiltenyiBiotech, Auburn, CA, USA). Cell suspensions were passedthrough a LSþ separation column attached to a MidiMACs sepa-ration unit and a magnet-retained fraction of purified CD34þ cellswas separated per manufacturer’s instructions. Clonogenic pro-genitor assays were performed in duplicate by plating 500 to1000 CD34þ selected cells in MethoCult H4434 semisolid me-dium (Stem Cell Technologies, Vancouver, BC, Canada). Differ-ent types of colony-forming units (CFUs) were morphologicallyscored after 14 days of incubation.

Enrichment, cell sorting,and expansion of hESC-derived CD73þ cellsFor enrichment of CD73þ cells, cocultured hESC/OP9 cells werecollected on day 8 using collagenase-IV and trypsin/EDTA andtransferred to new plates in a-MEM/10% FBS/NEAA/glu (mesen-chymal media). Three days later, when new culture dishes becamenear-confluent, cells were collected after treatment with trypsin/EDTA and analyzed by flow cytometry. CD73þ cells were sortedfrom this population of cells with FACSVantage SE with DiVa(BD Biosciences). CD73þ sorted cells were replated at a concen-tration of 5 � 105 cells/mL in mesenchymal media, and werepassaged into new culture dishes whenever they became near-confluent and analyzed by flow cytometry.

Osteogenic differentiation, vonKossa staining, and quantitative calcium assayFor osteogenic differentiation, CD73þ cells were grown in a-MEM/10% FBS media containing osteogenic supplements (0.1mM dexamethasone, 10 mM b-glycerol phosphate, and 200 mM as-corbic acid) [18,22] with medium change twice/week. Cell cul-tures were assayed for mineral content by the von Kossa method[23]. In brief, cell layers were rinsed with PBS, fixed with 10%formalin, incubated with 2% silver nitrate for 30 minutes under ul-traviolet light, washed, and counterstained with hematoxylin stain.For the quantitative calcium assay in the osteogenic-induced cul-ture, supernatants were processed according to manufacturer’s in-structions contained within the calcium quantification Sigma kit#587 (Sigma-Aldrich, St Louis, MO, USA). Absorbances fromthe samples were read at 575 nm. The calcium measurementswere calculated using standard solutions prepared in parallel andexpressed as ug/mL. Controls for both experiments included

148 P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

mesenchymal cells that were not induced with osteogenicsupplements.

Adipogenic differentiation, and Oil Red O stainingFor adipogenic differentiation, cells were grown in a-MEM/10%FBS media containing adipogenic supplement (1 mM dexametha-sone, 0.5 mM methyl-isobutylxanthine, and 10 U/mL insulin)[18,22] with media changes twice/week. Adipogenic differentia-tion was assessed through observation of the accumulation oflipid-rich vacuoles within cells after Oil Red O staining. Briefly,cells were rinsed, fixed with 10% formalin, rinsed again, and lay-ered with 60% isopropanol for 5 minutes. Then, isopropanol waspoured off and cells were stained with Oil Red O stain (Sigma-Aldrich), rinsed, counterstained with hematoxylin, rinsed again,and observed under phase-contrast microscopy. Controls includedmesenchymal cells that were not induced with adipogenicsupplements.

Chondrogenic differentiation and Safranin O staininghESC-derived mesenchymal cells were grown in a-MEM/10%FBS media containing chondrogenic supplements (10 ng/mLtransforming growth factor-b3 and 200 mM ascorbic acid) asa dense cell mass incubated at 37�C/5% CO2 in 15 mL conicaltubes with the caps slightly open [24]. Medium was changed every3 days without disturbing the cell mass. Cell sections were madeafter fixing the cell pellet with 10% formalin and embedding it inparaffin. The chondrogenic-induced cells and control cells (mes-enchymal cells not induced with chondrogenic supplements)were stained with Safranin O for glycosaminoglycans. Briefly,cells were deparaffinized in xylene and ethanol, stained with Wei-gert’s iron hematoxylin, and then destained with fresh acid alco-hol. Cells were then stained with 0.02% aqueous fast greenFCF, washed in 1% acetic acid, and stained with 0.1% aqueousSafranin O.

RT-PCR analysisWe used reverse transcriptase polymerase chain reaction (RT-PCR) using primers and conditions described previously [18,22],and visualized the products with agarose gel electrophoresis.Briefly, total RNA from the cultured cells was isolated using Tri-zol reagent, and 1 ug total RNA/each reaction was reverse tran-scribed with the Superscript III first-strand synthesis system(Invitrogen) to cDNA. PCR amplification was done with primersspecific to bone-specific protein (BSP-osteogenic specific), perox-isome proliferators-activated receptor g2 (PPARg2-adipogenicspecific), and type II collagen (chondrogenic specific). RT-PCRfor b-2 microglobulin was used as internal control in eachexperiment.

Results

Time course of emergence ofCD34þ and CD73þ cells in coculturesWe derived highly purified populations of human CD73þ

MSCs by the differentiation of undifferentiated hESCs(H1, H9, and GFPþ/H9-hESCs) through coculturing withirradiated OP9 cells. To study the time course for the ap-pearance of hematopoietic and mesenchymal cells in this

coculture system, we focused on the evaluation of the ex-pression of human-specific CD34 antigen (a marker ofprimitive human hematopoietic cells) and human-specificCD73 antigen (a marker of adult [22] or fetal [25] tissue-derived human MSCs) on the cultured cells, starting atday 4 of the cocultures. We consistently observed the emer-gence of CD34þ cells at day 5 of the coculture, shortly fol-lowed by the appearance of CD73þ cells. The time courseof appearance of CD34þ and CD73þ cells based on threesets of experiments are shown in (Fig. 1). We consistentlyobserved that in this coculture system, the temporal kineticsof CD73þ cells, including their peak and decline, followeda pattern similar to that of CD34þ cells. In contrast to theBarberi et al. study [18] in which CD73þ cells were iso-lated at day 40 of hESC/OP9 coculture, our cocultured cellswere difficult to sustain for more than 2 weeks, which waslikely because we always irradiated our OP9 cells prior tococulturing with hESCs. This was done mainly to preventproliferation of these cells in the culture and preventthem from surviving to further passages.

Purification of CD73þ MSCsderived from hESC/OP9 coculturesTo generate a pure population of CD73þ cells, hESC/OP9cocultured cells were collected on day 8 and replated intonew culture dishes in mesenchymal media. Three days laterwhen these cells became nearly confluent, the attachedmonolayers were collected and analyzed by flow cytometry.During the 3 days of enrichment, the percentage of CD73þ

cells increased significantly (p ! 0.001), from an averageof about 5% on day 8 to an average of 21% of the total cells3 days later (range, 18–24%) based on five different sets ofenrichment experiments (Fig. 2A, B). This increase in thepercentage of the CD73þ cells allowed us to collect a largeenough number of cells for additional purification throughfluorescent-activated cell sorting (FACS). After platingthe CD73þ cells that were sorted by FACS at the end of

Figure 1. Kinetics of appearance of CD34þ hematopoietic and CD73þ

mesenchymal cells in the first 2 weeks of human embryonic stem cells/

OP9 cocultures.

149P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

Figure 2. Purification of CD73þ cells. (A) Enrichment procedure: Approximately 5% of human embryonic stem cells/OP9 cocultured cells are positive for

CD73 surface antigen on day 8. Replating of the day 8 cells in mesenchymal media quadruples the percentage of CD73þ cells. (B) Flow cytometric analysis

of the replated cells at the end of 3 days of enrichment showing that 22% of the cells were CD73þ. These CD73þ cells were sorted by fluorescent-activated

cell sorting and cultured in mesenchymal media. (C) Morphology of CD73þ cells that were sorted at the end of enrichment and cultured in mesenchymal

media (photograph taken with Leica DFC320 digital camera on Leica DM IL inverted microscope with C Plan 10�/0.22 LMC objective). (D) Flow

cytometric analysis of sorted cells at the end of passage 1 shows that the majority of the cells are CD73þ and there is no hematopoietic CD34þ or

CD45þ cells left. PE 5 phycoerythrin.

the 3-day enrichment period, they exhibited spindle-shapedmorphology typical of adult bone marrow-derived mesen-chymal cells (Fig. 2C). Upon reaching confluency, thesecells were almost all CD73þ cells and, importantly, theywere negative for the hematopoietic cell surface markersCD34 and CD45 (Fig. 2D). Subsequent passages weredone whenever the cultured cells became near confluent.We were able to freeze, thaw, and subsequently passagethese CD73þ cells. Similar to the CD73þ MSCs thatwere not frozen prior to their differentiation assays, thecells that were frozen and then thawed maintained their dif-ferentiation potential into osteogenic, adipogenic, andchondrogenic lineages. We were able to keep CD73þ cellsderived in different experiments, on average, up to passage15 (range, 12–17), either continuously from the first platingafter FACS or based on the total number of passages pre-freeze and post-thaw. However, later passages of cells hada consistently slower growth rate and doubling time.

We observed similar kinetics in generation of CD34þ

and CD73þ cells when GFPþ/hESCs were coculturedwith OP9 cells. Using the same enrichment and sortingmethodology, we derived a pure population of GFPþ/CD73þ cells from our GFPþ/hESC line. These cells ex-hibited the same mesenchymal/fibroblast-looking morphol-

ogy (Fig. 3A); furthermore, they were positive for markersof bone marrow MSCs including CD73þ (97.6%), CD29þ

(99.6%), CD44þ (97.9%), CD54 (54.8%), CD90þ

(99.3%), CD105 (88.3%) (Fig. 3B), and negative for hema-topoietic markers CD34 and CD45. This pattern was simi-lar to the cell surface markers of our non-GFP/CD73þ

purified cells (data not shown) indicating that GFP expres-sion had no effect on the differentiation potential of GFPþ/hESCs toward MSCs in this system.

Hematopoietic colony-forming potentialof CD34þ cells derived from hESC/OP9 coculturesTo verify the hematopoietic progenitor potential of theCD34þ cells generated along CD73þ cells, we selectedCD34þ cells from cocultures of hESCs with OP9 cells atday 8 using the Miltenyi separation system. When theseCD34þ cells were plated in Methocult semi-solid mediathey generated colonies of different lineages verifyingthat the CD34þ cells generated along with the CD73þ cellsin these cocultures were indeed primitive hematopoieticcells as has been reported by other investigators [10,26].Two representative GFPþ colonies (one CFU-erythroidand one CFU-granulocyte macrophage) derived fromGFPþ/hESC cocultures with OP9 cells are shown in

150 P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

Figure 3. Purification of green fluorescent protein (GFP)þ/CD73þ cells. (A) Morphology of GFPþ/CD73þ mesenchymal cells derived from GFPþ/human

embryonic stem cells cocultured with murine OP9 cells after enrichment and sorting (photomicrograph taken with RT Slider camera on Olympus BXS1

microscope with UPlanFl 10�/0.30 objective, and GFP filter). (B) Flow cytometric analysis of cultured GFPþ/CD73þ cells for mesenchymal cell markers

at the end of passage 1. APC 5 allophycocyanin; PE 5 phycoerythrin.

(Fig. 4) to unequivocally verify the generation of hemato-poietic colonies from hESC-derived CD34þ cells; andagain verifying that the GFP expression had no effect onthe differentiation potential of hESCs.

Differentiation of hESC-derived MSCs towardosteogenic, adipogenic, and chondrogenic lineagesWe differentiated the MSCs of different passages afterCD73þ sorting (from passage 4 up to passage 9) toward os-

teogenic, adipogenic, and chondrogenic lineages by usingthe established methodologies. We used von Kossa stainingto demonstrate deposits of calcium crystals in the MSC cul-tures that were induced with osteogenic supplements. Apicture of typical brown-black calcium crystals (crystallinehydroxyapatite) is shown in (Fig. 5D). In one set of exper-iments, we used serial calcium deposition assays on days 4,7, 11, and 13 as a more accurate quantitative measure of thegeneration of calcium crystals in our culture system

151P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

Figure 4. Green fluorescent protein (GFP)þ colony-forming units (CFU) (A,C: CFU-erythroid; B,D: CFU-granulocyte macrophage) generated from CD34þ

cells that were selected from day 8 of GFPþ/human embryonic stem cells cocultured with OP9 cells. (Photographs taken with Leica DFC300FX digital

camera on Leica DM IL inverted microscope with C Plan 10�/0.22 LMC objective with and without GFP filter).

(Fig. 6). In the osteogenic-induced cultures, we noticeda progressive increase in the level of measured calciumfrom 6.5 ug/mL on day 4 to 23 ug/mL on day 13. However,in the control cultures (mesenchymal cells cultured in theabsence of osteogenic supplements), the calcium measure-ment remained within the background level of 0.2 to0.5 ug/mL.

Adipogenic differentiation was confirmed by the demon-stration of neutral lipid vacuoles by Oil Red O staining ofthe cells. In some experiments, we noticed evidence of adi-pogenic differentiation as early as 5 days after initiation ofthe adipogenic induction of MSCs with adipogenic supple-ments; a representative picture (Fig. 5E) shows that by day11, the majority of cells contained lipid vacuoles. No adipo-genic phenotypes were induced in the hESC-derived mes-enchymal cells that were cultured without adipogenicsupplements.

For assessment of the chondrogenic potential of ourhESC-derived MSCs, we cultured them as a cell pellet inthe micromass culture system in the presence of chondro-genic supplements. We verified chondrogenesis in the cellmass through Safranin O staining of the tissue sectionsfor detection of cartilage-specific glycosaminoglycans(Fig. 5F).

RT-PCR analysisWe used RT-PCR to further confirm generation of osteo-genic, adipogenic, and chondrogenic cells in our differenti-ation assays. Osteogenic cell generation was confirmed

using primers specific to BSP, adipogenic cells with primersspecific to PPARg2, and chondrogenic cells with primersspecific to type II collagen (Fig. 5G). We did not see corre-sponding bands in our control cells, hESC-derived CD73þ

cells, which were not induced with differentiationsupplements.

DiscussionDirected differentiation of hESCs into hematopoietic cells,either primitive or differentiated, provides a unique modelto study the developmental ontogeny of hematopoiesis invitro. Furthermore, if clinically acceptable methodologiesare developed, such hESC-derived hematopoietic cellscould potentially be used for transplantation/transfusionpurposes. However, hematopoietic stem cells derivedfrom hESCs could play a much bigger role in regenerativemedicine by providing a strategy for establishing tolerancein the recipient, through hematopoietic chimerism, towardother tissues (such as insulin-producing pancreatic cells)derived from the same hESC line.

Multipotent MSCs of adult origin, such as bone marrow-derived, are defined by a combination of morphologic,immunophenotypic, growth characteristics, and, most im-portantly, their differentiation potential into multiplemesenchymal lineages, including osteogenic, adipogenic,and chondrogenic cells [27–30]. More recently it hasbeen shown that, at least in some experimental models,

152 P. Trivedi and P. Hematti/ Experimental Hematology 35 (2007) 146–154

A B

F

-2 microglobulin

PPAR 2BSP Collagen-II

G

2

-OS +OS -AS +AS -CS +CS

Ind

uce

dC

on

tro

ls

C

E

D

Figure 5. Differentiation of mesenchymal cells toward osteogenic, adipogenic, and chondrogenic lineages. (A,D) von Kossa staining of mesenchymal cells

cultured in the absence (A) or presence (D) of osteogenic supplements. Deposit of calcium crystals are visualized as brown-black crystals in mesenchymal

cell cultures induced with osteogenic supplements for 21 days. (B,E) Oil Red O staining of the mesenchymal cells that were cultured without (B) or with (E)

adipogenic supplements show the presence of red lipid vacuoles in the majority of the latter cells by day 11. (C,F) Safranin O staining of the tissue sections

from day 17 of control (C) or chondrogenic-induced (D) mesenchymal cells grown as cell masses show the presence of glycosaminoglycans as red-orange

deposits in the latter. Photographs taken with Optronics camera on Leica DM IRB microscope with C Plan 10�/0.22 (A,B,D,E) and N plan 5�/0.12 (C,F)

objectives. (G) Reverse transcription polymerase chain reaction for bone-specific protein (BSP-osteogenic specific), peroxisome proliferators-activated

receptor g2 (PPARg2-adipogenic specific), and type II collagen (chondrogenic specific); images were taken using FOTODYNE imaging system with

Hamamatsu digital camera and Ethidium filter.

MSCs of adult origin can also contribute to regeneration ofnonmesenchymal tissues, including but not limited to, heart[31] and central nervous system [32] through a variety ofmechanisms [33]. MSCs have also been shown to possesother favorable characteristics, such as a lack of immunoge-nicity [34], which make them even more attractive as primecandidates for cell therapy applications.

Since the original description of the murine OP9 stro-mal cell line and its capability for supporting the generationof primitive hematopoietic cells from murine ESCs [12],this cell line has proven to be very useful for studying thegeneration of both primitive and more differentiated hema-topoietic cells from the ESCs of a variety of species includ-ing human ESCs [10–17,35–40]. Although Barberi et al.recently showed the emergence of a population of CD73þ

multipotent mesenchymal precursors comprising 5% of to-tal cells from day 40 of cocultured hESC/OP9 cells [18], wehypothesized that CD73þMSCs might appear earlier in thiscoculture system. No previous study has looked at the gen-eration of mesenchymal cells along with hematopoieticcells in this coculture system. Therefore, to characterizethe earliest stages of the development of CD73þ MSCs inthe hESC/OP9 coculture system, we specifically followed

the development of both CD34þ primitive hematopoieticcells and CD73þ MSCs in the first 2 weeks of cocultures.The kinetics of the generation of CD34þ cells from hESCcocultured with OP9 cells in our hands were similar towhat has been reported in other studies; furthermore theseCD34þ cells exhibited hematopoietic-colony formingpotential similar to what has been reported by other inves-tigators [10,15,26], indicating that these CD34þ cells areindeed primitive hematopoietic cells. We observed thatCD73þ cell generation in the first 2 weeks of coculturefollows a temporal pattern similar to that of CD34þ cells.We have also devised a two-step methodology to isolatea pure population of CD73þ cells. The enrichment processallows for quadrupling the number of CD73þ cells fromabout 5% on day 8 to about 21% in 3 days, at which pointsorting with FACS provides us with a pure population ofCD73þ cells. These cells had the typical morphology andcell surface marker expression characteristics similar toMSCs cultured from adult bone marrow samples. The exactcell surface antigenic phenotype of adult bone marrow-derived MSCs is still a matter of debate. Nevertheless,we showed that CD73þ cells generated from hESC/OP9cocultures could be differentiated into the osteogenic,

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adipogenic, and chondrogenic lineages, a hallmark propertyof MSC. Although we used the acronym MSC for ourhESC-derived mesenchymal cells, we have not shown yetthat these cells are capable of differentiation into multiplelineages at a clonal level [41].

Olivier et al. [42] have recently reported the generationof CD73þ mesenchymal cells from hESCs without cocul-turing with OP9 stromal cells. This methodology includeda complex multistep process that required more thana month of tissue culture. Based on the current experiments,we cannot prove if OP9 cells are indispensable for MSCgeneration, but it seems that they at least provide a milieuinductive to the early appearance of CD73þ cells alongwith CD34þ cells. It is also notable that our starting popu-lation of undifferentiated hESCs had been cultured on ma-trigel plates with mouse embryonic fibroblast-conditionedmedia for several passages instead of being directly main-tained on mouse embryonic fibroblasts prior to their differ-entiation inducing cultures. It has been previously shownthat hESCs cultured on matrigel with MEF-conditioned me-dia retain their hematopoietic development potential [5]and our experiments show that it does not interfere withthe CD73þ cell generation potential of hESCs either.

Cocultures of hESCs with OP9 stromal cells haveproven to be very valuable for the study of embryonic he-matopoiesis in vitro. To our knowledge, we have shownfor the first time the simultaneous appearance of both prim-itive hematopoietic cells and mesenchymal stem cells inthis coculture system, thus providing a useful in vitro meth-odology for studying the relationship between generation ofhematopoietic and mesenchymal cells. Furthermore, ourmethodology also provides a quick and efficient way to iso-late a pure population of CD73þMSCs. Through additionalrefinement and/or manipulation, hESC-derived MSCs couldpotentially provide an alternative to murine stromal celllines for supporting hematopoiesis from hESCs in vitro orthey could potentially be used to enhance engraftment of

0

5

10

15

20

25

0 2 5 8 10 11 12 13 14Days

with OScontrol

Cal

cium

ug/

ml

1 3 4 6 7 9

Figure 6. Quantitative measurement of calcium crystal formation in mes-

enchymal cells induced with osteogenic supplement (OS) vs control (no

OS) cultures.

hematopoietic cells derived from hESCs in vivo [43,44].This study provides additional evidence regarding the valueof the hESC/OP9 coculture methodology for studyinghESC-derived hematopoiesis.

AcknowledgmentsThis work was done in part through a grant from Trillium Fund forMultiple Myeloma Research at University of Wisconsin Paul P.Carbone Comprehensive Cancer Center. We are grateful to Dr.Yi Ping Liu for providing us with YPL2-EGFP plasmid, and toKathy Schell, Joel Puchalski, and Colleen Urben at the FlowCytometry Core Facility of the University of Wisconsin Paul P.Carbone Comprehensive Cancer Center for their FACS sorting.

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