Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.     

In vitro differentiation of mesenchymal progenitor cells derived from porcine umbilical cord blood

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.     

Identification and analysis of a novel human surface CD5- B lymphocyte subset producing natural antibodies.

The production of "natural" autoantibodies or antibodies, i.e., Ig that bind a variety of self- and/or exogenous Ag and arise independently of known immunization, is though to be a feature of CD5+ B lymphocytes. To determine whether other lymphocyte subsets exist that might be committed to the production of natural antibodies, human peripheral blood B cells were sorted on the basis of surface CD5 expression and differential expression of surface CD45RA (CD5+CD45RAintermediate(int), CD5-CD45RAlow(lo), and CD5-CD45RAhigh(hi)), and analyzed for the type of Ig produced after EBV infection and culture. Like their CD5+ counterparts, most CD5-CD45RAlo B lymphocytes were precursors of cells producing IgM, a major proportion of which displayed the Ag-binding features of natural antibodies. In contrast, CD5-CD45RAhi B cells comprised a high frequency of IgG-producing cell precursors, possibly including memory B lymphocytes. Six of seven IgM mAb generated from sorted CD5-CD45RAlo B cells and three of four IgM mAb from sorted CD5+ B cells were polyreactive, binding with different affinities (Kd, 10(-5) to 10(-8) M) to two or more Ag; the remaining mAb from CD5-CD45RAlo and the mAb from CD5+ B cells each bound to a single Ag (Kd, 10(-7) to 10(-8) M), beta-galactosidase and ssDNA, respectively. CD5-CD45RAlo B cells account for 4.1 +/- 1.2% (mean +/- SD in 11 healthy subjects; CD5+ B cells, 23.3 +/- 6.9%) of total B lymphocytes and display the features of quiescent cells. In a given individual, the number of CD5-CD45RAlo B cells remains constant over time. CD5-CD45RAlo and CD5+ B cells bear surface CD11b and CD14, at densities and/or frequencies apparently higher than those of CD5-CD45RAhi B lymphocytes. Despite their surface CD5- phenotype, CD45RAlo B cells express CD5+ mRNA at levels comparable with those of CD5+ B lymphocytes, whereas CD5-CD45RAhi B cells express only trace amounts of CD5 mRNA. The commitment to natural antibody production and the degree of CD5 mRNA expression suggest that the newly defined CD5-CD45RAlo B cell subset is related to CD5+ B lymphocytes, and may constitute the human homologue of the mouse Ly-1-"sister" B cell population.     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

Molecular cloning and chromosomal mapping of a candidate cytokine gene selectively expressed in human CD34+ cells

A rare population of human bone marrow (BM) and cord blood (CB) mononuclear cells bearing the CD34 surface marker (CD34+) function as hematopoietic stem/progenitor cells. Cells lacking CD34 expression (CD34-) in BM and CB are largely mature hematopoietic cells of various lineages that are derived from the CD34+ cells. To elucidate molecular mechanisms governing functional differences between CD34+ and CD34- hematopoietic cells, we used representational difference analysis (RDA)-based subtraction to identify genes that are specifically or preferentially expressed in CD34+ cells. Among the 73 RDA fragments initially sequenced, 30% are derived from the CD34 and c-kit genes that are preferentially expressed in CD34+ cells. An additional 27 (37%) are novel or homologous only to entries in expressed sequence tag databases. One (C17) was found four times and is expressed in CD34+ but not in CD34- cell populations from CB or BM. The cloned C17 cDNA encodes a novel polypeptide of 136 amino acids with a signal sequence. No homology to this peptide was found in the public databases. A secondary-structure analysis predicts that the C17 peptide contains four alpha-helices, a characteristic of hematopoietic cytokines and interleukins. This novel gene is mapped to human chromosome 4p15-p16.     

A multicolor,no-lyse no-wash assay for the absolute counting of CD34+ cells by flow cytometry

BACKGROUND: We previously developed a method for counting CD34(+) cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse-no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34(+) cells cannot be included. METHODS: We used SYTO-13 and fluorescein isothiocyanate (FITC)-CD45 staining (FL1) instead of SYTO-13 and phycoerythrin (PE)Cy5-CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34(+) cells. The new method was validated by applying it to cord blood samples (n = 20). RESULTS: FITC-CD45 antibody gave a 1.7-fold increase in mean fluorescence intensity over SYTO-13 alone. From absolute counts (CD34(+) cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges. CONCLUSIONS: No-lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249-253, 2002.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

Analysis of human TIE2 function on hematopoietic stem cells in umbilical cord blood

To investigate the behavior of hematopoietic stem cells (HSCs) in cord blood (CB), we analyzed the expression and function of TIE2, a tyrosine kinase receptor. A subpopulation of Lineage (Lin)(-/low)CD34(+) cells in CB expressed TIE2 (18.8%). Assays for long-term culture-initiating cells (LTC-IC) and cobble-stone formation revealed that Lin(-/low)CD34(+)TIE2(+) cells showed to have a capacity of primitive hematopoietic precursor cells in vitro. When Lin(-/low)CD34(+)TIE2(+) cells were cultured on the stromal cells, they transmigrated under the stromal layers and kept an immature character for a few weeks. By contrast, Lin(-/low)CD34(+)TIE2(-) cells differentiated immediately within a few weeks. Finally, we confirmed that 1x10(4)Lin(-/low)CD34(+)TIE2(+) cells were engrafted in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, while 1x10(4)Lin(-/low)CD34(+)TIE2(-) cells were not. Taken together, we conclude that TIE2 is a marker of HSCs in CB. A ligand for TIE2, Ang-1 promoted the adhesion of sorted primary Lin(-/low)CD34(+)TIE2(+) cells to fibronectin (FN), and this adhesion may play a critical role in keeping HSCs in an immature status under the stromal cells.     

HIV-gp120 induced cell death in hematopoietic progenitor CD34+ cells

Haematologic abnormalities accompany the majority of HIV-1 infections. At present it is unclear whether this is due directly to HIV infection of hematopoietic progenitor cells, or whether this results from an indirect mechanism secondary to HIV infection. Here we provide evidence for an indirect mechanism, whereby hematopoietic progenitor cells undergo HIV gp120-induced apoptosis (programmed cell death) even in the absence of HIV infection. Freshly isolated, purified human hematopoietic progenitor CD34+ cells, derived from both umbilical cord blood and bone marrow, co-expressed the CD4 marker at low density on their surface. Although these CD34+CD4+ cells theoretically should be capable of productive infection by HIV, we found that HIV-IIIB could not establish productive infection in these cells. Nonetheless, gp120 from IIIB could bind the cells. Thus, binding of gp120 did not correlate with infectivity. Furthermore, binding of gp120 was a specific event, leading to apoptosis upon crosslinking with anti-gp120 through a fas-dependent mechanism. If apoptosis is also observed in vivo even in uninfected hematopoietic cells, this could contribute significantly to the impairment in hematopoietic cell number and function. Our data suggest a novel indirect mechanism for depletion of CD34+ and CD34+-derived cells even in the absence of productive viral infection of these cells.     

Identification and analysis of in vitro cultured CD45-positive cells capable of multi-lineage differentiation

We report on a subset of cells that co-purify with CD45-positive/Lineage minus (CD45(pos)/Lin(minus)) hematopoietic cells that are capable of in vitro differentiation into multi-potential cells including cells with neuroectoderm properties. Although these cells are CD45 positive and have properties similar to CD45-negative mesenchymal progenitor cells (MPC) derived from bone marrow (BM), they are neither hematopoietic cells nor mesenchymal cells. These CD45(pos)/Lin(minus) cells can be expanded in vitro, express the stem cell genes Oct-4 and Nanog and can be induced to differentiate into endothelial cells, osteoblasts, muscle cells and neural cells at frequencies similar to those reported for bone marrow mesenchymal cells. Long-term culture of these cells followed by transplantation into NOD/SCID mice resulted in positive bone marrow stromal cell engraftment but not hematopoietic engraftment, suggesting that despite their CD45-positive status these cells do not have the same properties as hematopoietic stem cells. Clonal cell analysis determined that the culture period caused a broadening in the differentiation potential of the starting population.     

The human placenta is a hematopoietic organ during the embryonic and fetal periods of development

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations—CD34⁺⁺CD45^low and CD34⁺CD45^low—that were found in chorionic villi and in the chorioamniotic membrane. CD34⁺⁺CD45^low cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34⁺⁺CD45^low cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56⁺ natural killer cells and CD19⁺CD20⁺sIgM⁺ B cells in polyclonal liquid cultures. CD34⁺CD45^low cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34⁻ cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34⁺⁺CD45^low cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.     

BM cells giving rise to MSC in culture have a heterogeneous CD34 and CD45 phenotype

BACKGROUND: Mesenchymal stromal cells (MSC) isolated from adult human BM are characterized by their fibroblast-like morphology, adherent growth and capacity to differentiate into adipocytes, osteocytes, chondrocytes, cardiomyocytes and neuroprogenitors. After culturing these cells in vitro, they express the cell-surface molecules CD44, CD90, SH2 and SH3, and are negative for CD34 and the hematopoietic marker CD45. The aim of this study was to characterize the in vivo phenotype of MSC relative to the expression of CD34 and CD45. METHODS: BM mononuclear cells were stained with Ab against both molecules and separated into the CD34(+), CD34(-), CD45(+) CD34(+), CD45(high+) CD34(-), CD45(med,low+) CD34(-) and CD45(-) CD34(-) subpopulations, which were then cultured under the same conditions and analyzed for growth of MSC. RESULTS: A small population of MSC arose from the CD45(+) CD34(+) fraction, although the majority was obtained from the CD45(-) CD34(-) subpopulation. MSC from all fractions could be differentiated into adipocytes and osteocytes. In addition, MSC from the CD34(+) and CD34(-) fractions were shown to differentiate into chondrocytes. After in vitro culture, MSC from both fractions possessed the same phenotype, which was negative for CD34 and CD45. DISCUSSION: MSC from the CD45(+) CD34(+) fraction change their phenotype under in vitro conditions.     

Cutting edge: cytolytic effector function in human circulating CD8+ T cells closely correlates with CD56 surface expression

Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.     

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.     

Expanded CD34+ human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2rgamma(null) mice

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.     

Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.     

人造血干/祖细胞多态性的研究:Ⅲ.骨髓CD34~ 造血干/祖细胞功能亚群的分析

CD 34~ 造血干/祖细胞(HSC/HPC)是十分异质性的,由多个不同功能亚群所构成,在分化方向与重建造血等方面差异显著。本文对正常人骨髓CD 34~ HSC/HPC各亚群进行了较全面的分析。首先以阳性选择策略,采用Isolex~(TM) 50免疫磁球分选术富集骨髓CD 34~ HSC/HPC,其纯度>90%,随之采用免疫荧光抗体双标记二维流式细胞仪对其测定,发现高纯度的CD 34~ HSC/HPC可分为八个不同亚群:1.CD 34~ /CD 71~-(23.4%—56.6%)与CD 34~ /CD 71~ (33.4%—66.6%);2.CD34~ /CD 45~-(80.8%—82.5%)与CD34~ /CD 45~ (8.1%—11.2%);3.CD 34~ /CD 33~-(20.4%—80.6%)与CD 34~ /CD 33~ (14.6%—64.8%);4.CD 34~ /DR~-(6.3%—11.0%)与CD 34~ /DR~ (82.8%—85.5%)。用免疫胶体金——免疫桥酶联组化双染色对上述亚群进一步分析的结果与流式细胞仪的高度一致,为研究各亚群的功能与生物学特性提供了坚实基础     

Characterization of developmental pathway of natural killer cells from embryonic stem cells in vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(-) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(-) EB cells showed that CD45(+)Mac-1(-)Ter119(-) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(-)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(-) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(-) and they rapidly acquire CD122 as they differentiate along the NK lineage.