Simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells

Experimental Hematology 35 (2007) 146–154

Simultaneous generation of CD34þ primitive hematopoietic cells and CD73þ mesenchymal stem cells from human embryonic stem cells cocultured with murine OP9 stromal cells Parul Trivedia and Peiman Hemattia,b a Department 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+ primitive hematopoietic cells after several days of coculturing with the OP9 murine stromal cell line. CD73+ multipotent mesenchymal cells have also been isolated from hESC/OP9 cocultures after several weeks. We hypothesized that generation of CD34+ hematopoietic cells and CD73+ mesenchymal stem cells (MSCs) may follow similar kinetics, so we investigated the generation of CD73+ cells in the first 2 weeks of hESC/OP9 cocultures, at a time when CD34+ cells are generated. Materials and Methods. We cocultured hESCs with OP9 cells and examined the time course of appearance of human CD34+ and CD73+ cells using flow cytometry. We tested the hematopoietic progenitor potentials of CD34+ cells generated using hematopoietic colony-forming assays, and the multipotent mesenchymal properties of CD73+ cells generated using in vitro differentiation assays. Results. We observed that in the first 2 weeks of the hESC/OP9 coculture system CD34+ hematopoietic and CD73+ MSC generation follows a similar pattern. We sorted the CD34+ cells and showed that they can generate hematopoietic progenitor colonies. Starting with cocultured cells on day 8, and through an enrichment procedure, we also could generate a pure population of MSCs. These hESC-derived MSCs had typical morphological and cell surface marker characteristics of adult bone marrow-derived MSCs, and could be differentiated toward osteogenic, adipogenic, and chondrogenic cells in vitro, a hallmark property of MSCs. Conclusions. OP9 cells when cocultured with hESCs support simultaneous generation of CD34+ primitive hematopoietic cells and CD73+ MSCs from hESCs. Ó 2007 International Society 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 exponential interest in the potential use of these cells in regenerative medicine. However, before any human therapeutic applications can be achieved, there must be reproducible, efficient, and safe methodologies for directed differentiation of hESCs into desired cell types, either in vitro or in vivo. The derivation of primitive or differentiated blood cells, 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: [email protected]

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

0301-472X/06 $–see front matter. Copyright Ó 2007 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2006.09.003

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M-CSF. More recently, the OP9 cell line has also been used for efficient derivation of primitive hematopoietic cells from hESCs [10,15], as well as from nonhuman primate ESCs [16,17]. In this experimental system, CD34þ cells are usually generated from ESCs after several days of coculturing with OP9 cells, and then decline by 2 weeks. Interestingly, Barberi et al. [18] recently reported that 40 days after coculturing hESCs with OP9 cells an average of 5% of cocultured cells were CD73þ cells of human origin [18]. These cells were sorted, expanded, and shown to be multipotent mesenchymal precursors by their capability to differentiate into multiple mesenchymal derivatives, such as osteogenic, adipogenic, and chondrogenic cells. Because of the close ontogenic relationship between hematopoietic and their supportive mesenchymal stem (or stromal) cells (MSCs) [19,20], we hypothesized that these two types of cells might follow a similar pattern of generation in the hESC/OP9 coculture system. In this study, we investigated and characterized the kinetics of generation of primitive hematopoietic cells and MSCs from hESCs cocultured with OP9 cells.

Material and methods hESC and OP9 cell cultures The hESC cell lines H1 and H9 (federally registered as WA01 and WA09), passages 25–35 and karyotypically normal were obtained from WiCell (Madison, WI, USA), and maintained in an undifferentiated state by passaging on matrigel plates in mouse embryonic fibroblast (MEF)-conditioned media. New hESC lines expressing green fluorescent protein (GFP) were derived using the plasmid and methodology described by Liu et al. [21]. The OP9 cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and was maintained on gelatinized plates in amodified minimum essential (a-MEM) medium (Invitrogen, Carlsbad, CA, USA) containing 20% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 0.1 mM nonessential amino acid (NEAA), and 2 mM L-glutamine (glu). Coculture differentiation toward hematopoietic and mesenchymal cells Differentiation of hESCs toward hematopoietic and mesenchymal cells was induced by plating hESCs at a density of 1  105 cells mL onto six-well plates containing a confluent monolayer of OP9 cells irradiated with 80 Gy, and differentiation medium containing a-MEM/10% FBS/NEAA/glu and 0.1 mM b-mercaptoethanol. Cocultured cells were then incubated at 37 C/5% CO2 with half medium change on days 4, 6, and 8. Flow cytometry For flow cytometry, cocultured hESC/OP9 cells were collected at different time points after treatment with collagenase-IV (Invitrogen) followed by 0.05% trypsin/0.5 mM ethylenediamine tetraacetic acid (EDTA); similarly, mesenchymal cells generated later were collected after treatment with trypsin/EDTA. Dissociated cells were centrifuged and washed with phosphate-buffered saline (PBS) supplemented with 2% FBS and 0.1% sodium azide, and

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single-cell suspensions were prepared. Only human specific monoclonal antibodies tested to be nonreactive to OP9 cells were used for labeling of single-cell suspensions, including CD73 (IgG1, phycoerythrin [PE]), CD29 (IgG1, PE), CD34 (IgG1, PE), CD44 (IgG2b, PE), CD45 (IgG1, PE), CD54 (IgG1, PE), CD90 (IgG1, allophycocyanin) and CD105 (IgG1, allophycocyanin) (all from BD Biosciences (San Jose, CA, USA). Control staining, with appropriate isotype-matched monoclonal antibodies along with unstained control samples, was included in each experiment. Samples were analyzed using a FACSCalibur flow cytometer with Cell Quest acquisition software (BD Biosciences). List mode files were analyzed by FlowJo software (Tree Star, Ashland, OR, USA). CD34þ cell sorting and hematopoietic colony-forming cell assays Single-cell suspensions from day 8 of cocultures of hESCs with OP9 cells were labeled with CD34 paramagnetic monoclonal antibodies using Direct CD34 Progenitor Cell Isolation kit (Miltenyi Biotech, Auburn, CA, USA). Cell suspensions were passed through a LSþ separation column attached to a MidiMACs separation unit and a magnet-retained fraction of purified CD34þ cells was separated per manufacturer’s instructions. Clonogenic progenitor assays were performed in duplicate by plating 500 to 1000 CD34þ selected cells in MethoCult H4434 semisolid medium (Stem Cell Technologies, Vancouver, BC, Canada). Different types of colony-forming units (CFUs) were morphologically scored after 14 days of incubation. Enrichment, cell sorting, and expansion of hESC-derived CD73þ cells For enrichment of CD73þ cells, cocultured hESC/OP9 cells were collected on day 8 using collagenase-IV and trypsin/EDTA and transferred to new plates in a-MEM/10% FBS/NEAA/glu (mesenchymal media). Three days later, when new culture dishes became near-confluent, cells were collected after treatment with trypsin/ EDTA and analyzed by flow cytometry. CD73þ cells were sorted from this population of cells with FACSVantage SE with DiVa (BD Biosciences). CD73þ sorted cells were replated at a concentration of 5  105 cells/mL in mesenchymal media, and were passaged into new culture dishes whenever they became nearconfluent and analyzed by flow cytometry. Osteogenic differentiation, von Kossa staining, and quantitative calcium assay For osteogenic differentiation, CD73þ cells were grown in aMEM/10% FBS media containing osteogenic supplements (0.1 mM dexamethasone, 10 mM b-glycerol phosphate, and 200 mM ascorbic acid) [18,22] with medium change twice/week. Cell cultures 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 ultraviolet light, washed, and counterstained with hematoxylin stain. For the quantitative calcium assay in the osteogenic-induced culture, supernatants were processed according to manufacturer’s instructions contained within the calcium quantification Sigma kit #587 (Sigma-Aldrich, St Louis, MO, USA). Absorbances from the samples were read at 575 nm. The calcium measurements were calculated using standard solutions prepared in parallel and expressed as ug/mL. Controls for both experiments included

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mesenchymal cells that were not induced with osteogenic supplements. Adipogenic differentiation, and Oil Red O staining For adipogenic differentiation, cells were grown in a-MEM/10% FBS media containing adipogenic supplement (1 mM dexamethasone, 0.5 mM methyl-isobutylxanthine, and 10 U/mL insulin) [18,22] with media changes twice/week. Adipogenic differentiation was assessed through observation of the accumulation of lipid-rich vacuoles within cells after Oil Red O staining. Briefly, cells were rinsed, fixed with 10% formalin, rinsed again, and layered with 60% isopropanol for 5 minutes. Then, isopropanol was poured off and cells were stained with Oil Red O stain (SigmaAldrich), rinsed, counterstained with hematoxylin, rinsed again, and observed under phase-contrast microscopy. Controls included mesenchymal cells that were not induced with adipogenic supplements. Chondrogenic differentiation and Safranin O staining hESC-derived mesenchymal cells were grown in a-MEM/10% FBS media containing chondrogenic supplements (10 ng/mL transforming growth factor-b3 and 200 mM ascorbic acid) as a dense cell mass incubated at 37 C/5% CO2 in 15 mL conical tubes with the caps slightly open [24]. Medium was changed every 3 days without disturbing the cell mass. Cell sections were made after fixing the cell pellet with 10% formalin and embedding it in paraffin. The chondrogenic-induced cells and control cells (mesenchymal cells not induced with chondrogenic supplements) were stained with Safranin O for glycosaminoglycans. Briefly, cells were deparaffinized in xylene and ethanol, stained with Weigert’s iron hematoxylin, and then destained with fresh acid alcohol. Cells were then stained with 0.02% aqueous fast green FCF, washed in 1% acetic acid, and stained with 0.1% aqueous Safranin O. RT-PCR analysis We used reverse transcriptase polymerase chain reaction (RTPCR) 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 Trizol reagent, and 1 ug total RNA/each reaction was reverse transcribed with the Superscript III first-strand synthesis system (Invitrogen) to cDNA. PCR amplification was done with primers specific to bone-specific protein (BSP-osteogenic specific), peroxisome proliferators-activated receptor g2 (PPARg2-adipogenic specific), and type II collagen (chondrogenic specific). RT-PCR for b-2 microglobulin was used as internal control in each experiment.

coculture system, we focused on the evaluation of the expression of human-specific CD34 antigen (a marker of primitive human hematopoietic cells) and human-specific CD73 antigen (a marker of adult [22] or fetal [25] tissuederived human MSCs) on the cultured cells, starting at day 4 of the cocultures. We consistently observed the emergence of CD34þ cells at day 5 of the coculture, shortly followed by the appearance of CD73þ cells. The time course of appearance of CD34þ and CD73þ cells based on three sets of experiments are shown in (Fig. 1). We consistently observed that in this coculture system, the temporal kinetics of CD73þ cells, including their peak and decline, followed a pattern similar to that of CD34þ cells. In contrast to the Barberi et al. study [18] in which CD73þ cells were isolated at day 40 of hESC/OP9 coculture, our cocultured cells were difficult to sustain for more than 2 weeks, which was likely because we always irradiated our OP9 cells prior to coculturing with hESCs. This was done mainly to prevent proliferation of these cells in the culture and prevent them from surviving to further passages. Purification of CD73þ MSCs derived from hESC/OP9 cocultures To generate a pure population of CD73þ cells, hESC/OP9 cocultured cells were collected on day 8 and replated into new culture dishes in mesenchymal media. Three days later when these cells became nearly confluent, the attached monolayers 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 average of about 5% on day 8 to an average of 21% of the total cells 3 days later (range, 18–24%) based on five different sets of enrichment experiments (Fig. 2A, B). This increase in the percentage of the CD73þ cells allowed us to collect a large enough number of cells for additional purification through fluorescent-activated cell sorting (FACS). After plating the CD73þ cells that were sorted by FACS at the end of

Results Time course of emergence of CD34þ and CD73þ cells in cocultures We derived highly purified populations of human CD73þ MSCs by the differentiation of undifferentiated hESCs (H1, H9, and GFPþ/H9-hESCs) through coculturing with irradiated OP9 cells. To study the time course for the appearance of hematopoietic and mesenchymal cells in this

Figure 1. Kinetics of appearance of CD34þ hematopoietic and CD73þ mesenchymal cells in the first 2 weeks of human embryonic stem cells/ OP9 cocultures.

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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-shaped morphology typical of adult bone marrow-derived mesenchymal cells (Fig. 2C). Upon reaching confluency, these cells were almost all CD73þ cells and, importantly, they were negative for the hematopoietic cell surface markers CD34 and CD45 (Fig. 2D). Subsequent passages were done whenever the cultured cells became near confluent. We were able to freeze, thaw, and subsequently passage these CD73þ cells. Similar to the CD73þ MSCs that were not frozen prior to their differentiation assays, the cells that were frozen and then thawed maintained their differentiation potential into osteogenic, adipogenic, and chondrogenic lineages. We were able to keep CD73þ cells derived in different experiments, on average, up to passage 15 (range, 12–17), either continuously from the first plating after FACS or based on the total number of passages prefreeze and post-thaw. However, later passages of cells had a consistently slower growth rate and doubling time. We observed similar kinetics in generation of CD34þ and CD73þ cells when GFPþ/hESCs were cocultured with OP9 cells. Using the same enrichment and sorting methodology, we derived a pure population of GFPþ/ CD73þ cells from our GFPþ/hESC line. These cells exhibited the same mesenchymal/fibroblast-looking morphol-

ogy (Fig. 3A); furthermore, they were positive for markers of 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 hematopoietic markers CD34 and CD45. This pattern was similar to the cell surface markers of our non-GFP/CD73þ purified cells (data not shown) indicating that GFP expression had no effect on the differentiation potential of GFPþ/ hESCs toward MSCs in this system. Hematopoietic colony-forming potential of CD34þ cells derived from hESC/OP9 cocultures To verify the hematopoietic progenitor potential of the CD34þ cells generated along CD73þ cells, we selected CD34þ cells from cocultures of hESCs with OP9 cells at day 8 using the Miltenyi separation system. When these CD34þ cells were plated in Methocult semi-solid media they generated colonies of different lineages verifying that the CD34þ cells generated along with the CD73þ cells in these cocultures were indeed primitive hematopoietic cells as has been reported by other investigators [10,26]. Two representative GFPþ colonies (one CFU-erythroid and one CFU-granulocyte macrophage) derived from GFPþ/hESC cocultures with OP9 cells are shown in

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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 hematopoietic colonies from hESC-derived CD34þ cells; and again verifying that the GFP expression had no effect on the differentiation potential of hESCs. Differentiation of hESC-derived MSCs toward osteogenic, adipogenic, and chondrogenic lineages We differentiated the MSCs of different passages after CD73þ sorting (from passage 4 up to passage 9) toward os-

teogenic, adipogenic, and chondrogenic lineages by using the established methodologies. We used von Kossa staining to demonstrate deposits of calcium crystals in the MSC cultures that were induced with osteogenic supplements. A picture of typical brown-black calcium crystals (crystalline hydroxyapatite) is shown in (Fig. 5D). In one set of experiments, we used serial calcium deposition assays on days 4, 7, 11, and 13 as a more accurate quantitative measure of the generation of calcium crystals in our culture system

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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 noticed a progressive increase in the level of measured calcium from 6.5 ug/mL on day 4 to 23 ug/mL on day 13. However, in the control cultures (mesenchymal cells cultured in the absence of osteogenic supplements), the calcium measurement remained within the background level of 0.2 to 0.5 ug/mL. Adipogenic differentiation was confirmed by the demonstration of neutral lipid vacuoles by Oil Red O staining of the cells. In some experiments, we noticed evidence of adipogenic differentiation as early as 5 days after initiation of the adipogenic induction of MSCs with adipogenic supplements; a representative picture (Fig. 5E) shows that by day 11, the majority of cells contained lipid vacuoles. No adipogenic phenotypes were induced in the hESC-derived mesenchymal cells that were cultured without adipogenic supplements. For assessment of the chondrogenic potential of our hESC-derived MSCs, we cultured them as a cell pellet in the micromass culture system in the presence of chondrogenic supplements. We verified chondrogenesis in the cell mass through Safranin O staining of the tissue sections for detection of cartilage-specific glycosaminoglycans (Fig. 5F). RT-PCR analysis We used RT-PCR to further confirm generation of osteogenic, adipogenic, and chondrogenic cells in our differentiation assays. Osteogenic cell generation was confirmed

using primers specific to BSP, adipogenic cells with primers specific to PPARg2, and chondrogenic cells with primers specific to type II collagen (Fig. 5G). We did not see corresponding bands in our control cells, hESC-derived CD73þ cells, which were not induced with differentiation supplements.

Discussion Directed differentiation of hESCs into hematopoietic cells, either primitive or differentiated, provides a unique model to study the developmental ontogeny of hematopoiesis in vitro. Furthermore, if clinically acceptable methodologies are developed, such hESC-derived hematopoietic cells could potentially be used for transplantation/transfusion purposes. However, hematopoietic stem cells derived from hESCs could play a much bigger role in regenerative medicine by providing a strategy for establishing tolerance in the recipient, through hematopoietic chimerism, toward other tissues (such as insulin-producing pancreatic cells) derived from the same hESC line. Multipotent MSCs of adult origin, such as bone marrowderived, are defined by a combination of morphologic, immunophenotypic, growth characteristics, and, most importantly, their differentiation potential into multiple mesenchymal lineages, including osteogenic, adipogenic, and chondrogenic cells [27–30]. More recently it has been shown that, at least in some experimental models,

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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 of nonmesenchymal tissues, including but not limited to, heart [31] and central nervous system [32] through a variety of mechanisms [33]. MSCs have also been shown to posses other favorable characteristics, such as a lack of immunogenicity [34], which make them even more attractive as prime candidates for cell therapy applications. Since the original description of the murine OP9 stromal cell line and its capability for supporting the generation of primitive hematopoietic cells from murine ESCs [12], this cell line has proven to be very useful for studying the generation of both primitive and more differentiated hematopoietic cells from the ESCs of a variety of species including 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 total cells from day 40 of cocultured hESC/OP9 cells [18], we hypothesized that CD73þ MSCs might appear earlier in this coculture system. No previous study has looked at the generation of mesenchymal cells along with hematopoietic cells in this coculture system. Therefore, to characterize the earliest stages of the development of CD73þ MSCs in the hESC/OP9 coculture system, we specifically followed

the development of both CD34þ primitive hematopoietic cells and CD73þ MSCs in the first 2 weeks of cocultures. The kinetics of the generation of CD34þ cells from hESC cocultured with OP9 cells in our hands were similar to what has been reported in other studies; furthermore these CD34þ cells exhibited hematopoietic-colony forming potential similar to what has been reported by other investigators [10,15,26], indicating that these CD34þ cells are indeed primitive hematopoietic cells. We observed that CD73þ cell generation in the first 2 weeks of coculture follows a temporal pattern similar to that of CD34þ cells. We have also devised a two-step methodology to isolate a pure population of CD73þ cells. The enrichment process allows for quadrupling the number of CD73þ cells from about 5% on day 8 to about 21% in 3 days, at which point sorting with FACS provides us with a pure population of CD73þ cells. These cells had the typical morphology and cell surface marker expression characteristics similar to MSCs cultured from adult bone marrow samples. The exact cell surface antigenic phenotype of adult bone marrowderived MSCs is still a matter of debate. Nevertheless, we showed that CD73þ cells generated from hESC/OP9 cocultures could be differentiated into the osteogenic,

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

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This work was done in part through a grant from Trillium Fund for Multiple 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 to Kathy Schell, Joel Puchalski, and Colleen Urben at the Flow Cytometry Core Facility of the University of Wisconsin Paul P. Carbone Comprehensive Cancer Center for their FACS sorting.

Figure 6. Quantitative measurement of calcium crystal formation in mesenchymal cells induced with osteogenic supplement (OS) vs control (no OS) cultures.

References adipogenic, and chondrogenic lineages, a hallmark property of MSC. Although we used the acronym MSC for our hESC-derived mesenchymal cells, we have not shown yet that these cells are capable of differentiation into multiple lineages at a clonal level [41]. Olivier et al. [42] have recently reported the generation of CD73þ mesenchymal cells from hESCs without coculturing with OP9 stromal cells. This methodology included a complex multistep process that required more than a month of tissue culture. Based on the current experiments, we cannot prove if OP9 cells are indispensable for MSC generation, but it seems that they at least provide a milieu inductive to the early appearance of CD73þ cells along with CD34þ cells. It is also notable that our starting population of undifferentiated hESCs had been cultured on matrigel plates with mouse embryonic fibroblast-conditioned media for several passages instead of being directly maintained on mouse embryonic fibroblasts prior to their differentiation inducing cultures. It has been previously shown that hESCs cultured on matrigel with MEF-conditioned media retain their hematopoietic development potential [5] and our experiments show that it does not interfere with the CD73þ cell generation potential of hESCs either. Cocultures of hESCs with OP9 stromal cells have proven to be very valuable for the study of embryonic hematopoiesis in vitro. To our knowledge, we have shown for the first time the simultaneous appearance of both primitive hematopoietic cells and mesenchymal stem cells in this coculture system, thus providing a useful in vitro methodology for studying the relationship between generation of hematopoietic and mesenchymal cells. Furthermore, our methodology also provides a quick and efficient way to isolate a pure population of CD73þ MSCs. Through additional refinement and/or manipulation, hESC-derived MSCs could potentially provide an alternative to murine stromal cell lines for supporting hematopoiesis from hESCs in vitro or they could potentially be used to enhance engraftment of

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