Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors

Emerging data suggest that a subset of circulating human CD34(+) cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 +/- 0.3% of CD34(+) cells isolated from fetal liver (FL), 2 +/- 0.5% from mobilized peripheral blood, and 1.4 +/- 0.5% from cord blood were VEGFR-2(+). In addition, most CD34(+)VEGFR-2(+) cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34(+) cells phenotypically identifies a unique population of CEPs. CD34(+)VEGFR-2(+) cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34(+)VEGFR-2(+) cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34(+) cells that give rise to endothelial colonies, CD34(+) cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2(+) cells with VEGF and FGF-2 resulted in differentiation of AC133(+ )VEGFR-2(+) cells into adherent AC133(-)VEGFR-2(+)Ac-LDL(+ )(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133(+)VEGFR-2(+) cells. These data suggest that circulating CD34(+) cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.     

Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133(+) bone marrow cells, a subset of CD34(+) haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 +/- 5%), the CD133(+) bone marrow cells were grown on fibronectin-coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF-1). The CD133(+) fraction contained 95 +/- 4% CD34(+) cells, 3 +/- 2% cells expressing VEGF receptor (VEGFR-2/KDR), but did not express von Willebrand factor (VWF), VE-cadherin, P1H12 or TE-7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 +/- 5 times. The cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45(-) and CD14(-), and expressed several endothelial markers (UEA-1, VWF, P1H12, CD105, E-selectin, VCAM-1 and VE-cadherin) and typical Weibel-Palade bodies. They had a high proliferative potential (up to a 2400-fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co-cultivated with CD34(+) cells, in parallel with a purified fibroblastic cell monolayer. CD34(+) cells (10 x 10(5)) gave rise to 17,951 +/- 2422 CFU-GM colonies when grown on endothelial cells, and to 12,928 +/- 4415 CFU-GM colonies on fibroblast monolayers. The ECs also supported erythroid blast-forming unit (BFU-E) colonies better. These results suggest that bone marrow CD133(+) progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo-angiogenesis.     

Isolation and characterization of CD133+CD34+VEGFR-2+CD45- fetal endothelial cells from human term placenta

The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.     

Differentiation of endothelial cells from human umbilical cord blood AC133−CD14+ cells

Endothelial progenitor cells (EPCs) participate in neovascularization and are consistent with postnatal vasculogenesis. In vitro, they differentiate into endothelial cells (ECs). Prior reports have suggested that circulating human AC133(+) cells have the capacity to differentiate into ECs as progenitor cells. However, recent studies have demonstrated that circulating CD34(-)CD14(+) cells also have EPC-like properties in vitro and in vivo. We tested whether AC133(-)CD14(+) cells from human umbilical cord blood (HUCB) have the potential to differentiate into ECs. The AC133(-)CD14(+) cells were isolated from HUCB by magnetic bead selection and cultured on fibronectin-coated six-well trays in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and insulin growth factor (IGF-1). The AC133(-)CD14(+) cells adhered slightly within 1 day of culture and subsequently underwent a distinct process of morphological transformation to spindle-shaped cells that sprouted from the edge of the cell clusters. After 14 days, the cells formed cord- and tubular-like structures. The AC133(-)CD14(+) cells showed a strong increase in the endothelial marker P1H12 over time, whereas CD14 decreased, and CD45 did not change, respectively. In addition, the cells expressed endothelial markers von Willebrand's factor (vWF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial growth factor receptor-1 (VEGFR-1)/Flt-1, VEGFR-2/Flk-1, eNOS, and VE-cadherin, but did not express Tie-2 after 7 days of culture. The present data indicate that AC133(-)CD14(+) cells from HUCB are able to develop endothelial phenotype with expression of endothelial-specific surface markers and even form cord- and tubular-like structures in vitro as progenitor cells.     

Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells

Recent experiments show that hematopoietic progenitor cell populations contain endothelial precursor cells. We have isolated a population of CD34(+) cells that expresses fibroblast growth factor receptor-1 (FGFR-1) and that differentiates into endothelial cells in vitro. We find that 4.5% +/- 2.1% of CD34(+) cells isolated from bone marrow, cord blood, and mobilized peripheral blood express FGFR-1 and that viable CD34(+)FGFR(+) cells are small, with little granularity, and express both primitive hematopoietic and endothelial markers on their surface. The primitive hematopoietic markers AC133, c-kit, and Thy-1 are coexpressed by 75%, 85%, and 64% of CD34(+)FGFR(+) cells, respectively. Most of the CD34(+)FGFR(+) cells also express antigens found on endothelial cells, such as CD31, vascular endothelial growth factor receptor-2, and the endothelial-specific cell surface marker, vascular endothelial cadherin (VE-cadherin), whereas 56% to 60% of the cells express Tie, Tek, and the endothelial-specific marker, P1H12. The CD34(+)FGFR(+) population is enriched in cells expressing endothelial-specific antigens compared with the CD34(+) population. Isolated CD34(+)FGFR(+) cells grow slowly in culture, are stimulated by fibroblast growth factor-2 and vascular endothelial growth factor, and give rise to cells that express von Willebrand factor and VE-cadherin and that incorporate acetylated low-density lipoprotein. These experiments show that FGFR-1 is expressed by a subpopulation of CD34(+) cells that give rise to endothelial cells in vitro, indicating that this population contains endothelial stem/progenitor cells.     

Human CD34+AC133+VEGFR-2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors

OBJECTIVE: Endothelial progenitor cells (EPCs) are used for angiogenic therapies or as biomarkers to assess cardiovascular disease risk. However, there is no uniform definition of an EPC, which confounds EPC studies. EPCs are widely described as cells that coexpress the cell-surface antigens CD34, AC133, and vascular endothelial growth factor receptor-2 (VEGFR-2). These antigens are also expressed on primitive hematopoietic progenitor cells (HPCs). Remarkably, despite their original identification, CD34+AC133+VEGFR-2+ cells have never been isolated and simultaneously plated in hematopoietic and endothelial cell (EC) clonogenic assays to assess the identity of their clonal progeny, which are presumably the cellular participants in vascular regeneration. METHODS: CD34+AC133+VEGFR-2+ cells were isolated from human umbilical cord blood (CB) or granulocyte colony-stimulating factor-mobilized peripheral blood and assayed for either EPCs or HPCs. RESULTS: CD34+AC133+VEGFR-2+ cells did not form EPCs and were devoid of vessel forming activity. However, CD34+AC133+VEGFR-2+ cells formed HPCs and expressed the hematopoietic lineage-specific antigen, CD45. We next tested whether EPCs could be separated from HPCs by immunoselection for CD34 and CD45. CD34+CD45+ cells formed HPCs but not EPCs, while CD34+CD45- cells formed EPCs but not HPCs. CONCLUSIONS: Therefore, CD34+AC133+VEGFR-2+ cells are HPCs that do not yield EC progeny, and the biological mechanism for their correlation with cardiovascular disease needs to be reexamined.     

In vitro differentiation of endothelial cells from AC133-positive progenitor cells

Recent findings support the hypothesis that the CD34(+)-cell population in bone marrow and peripheral blood contains hematopoietic and endothelial progenitor and stem cells. In this study, we report that human AC133(+) cells from granulocyte colony-stimulating factor-mobilized peripheral blood have the capacity to differentiate into endothelial cells (ECs). When cultured in the presence of vascular endothelial growth factor (VEGF) and the novel cytokine stem cell growth factor (SCGF), AC133(+) progenitors generate both adherent and proliferating nonadherent cells. Phenotypic analysis of the cells within the adherent population reveals that the majority display endothelial features, including the expression of KDR, Tie-2, Ulex europaeus agglutinin-1, and von Willebrand factor. Electron microscopic studies of these cells show structures compatible with Weibel-Palade bodies that are found exclusively in vascular endothelium. AC133-derived nonadherent cells give rise to both hematopoietic and endothelial colonies in semisolid medium. On transfer to fresh liquid culture with VEGF and SCGF, nonadherent cells again produce an adherent and a nonadherent population. In mice with severe combined immunodeficiency, AC133-derived cells form new blood vessels in vivo when injected subcutaneously together with A549 lung cancer cells. These data indicate that the AC133(+)-cell population consists of progenitor and stem cells not only with hematopoietic potential but also with the capacity to differentiate into ECs. Whether these hematopoietic and endothelial progenitors develop from a common precursor, the hemangioblast will be studied at the single-cell level.     

Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting

Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in?vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM?+?5%, FCS?+?5% PRGF and IMDM?+?10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.     

Endothelial progenitor cells in infantile hemangioma

Infantile hemangioma is an endothelial tumor that grows rapidly after birth but slowly regresses during early childhood. Initial proliferation of hemangioma is characterized by clonal expansion of endothelial cells (ECs) and neovascularization. Here, we demonstrated mRNA encoding CD133-2, an important marker for endothelial progenitor cells (EPCs), predominantly in proliferating but not involuting or involuted hemangioma. Progenitor cells coexpressing CD133 and CD34 were detected by flow cytometry in 11 of 12 proliferating hemangioma specimens from children 3 to 24 months of age. Furthermore, in 4 proliferating hemangiomas, we showed that 0.14% to 1.6% of CD45(-) nucleated cells were EPCs that coexpressed CD133 and the EC marker KDR. This finding is consistent with the presence of KDR(+) immature ECs in proliferating hemangioma. Our results suggest that EPCs contribute to the early growth of hemangioma. To our knowledge, this is the first study to show direct evidence of EPCs in a human vascular tumor.     

CD34-/CD133+/VEGFR-2+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities

Our goal was to identify functionally important subpopulations within the heterogenous group of endothelial progenitor cells (EPC). Fluorescence-activated cell sorter analysis of CD133+ progenitor cells revealed the presence of CD34+ and CD34- subpopulations. CD34-/133+ progenitors differentiate into CD34+/133+ EPC, adhere more potently than these in response to SDF-1, and rapidly home to sites of limb ischemia in human volunteers. In human coronary atherectomy samples, fewer CD34-/133+ than CD34+/133+ EPC are present in stable plaques, whereas cell numbers increase with a reversion of the ratio in unstable lesions. In CD34-/133+ EPC-injected nude mice, more transplanted cells coexpressing endothelial markers home to carotid artery lesion endothelium than in CD34+/133+-injected mice. In the former, lesions were smaller and reendothelialization higher than in the latter. We identified a new CD34-/133+ EPC subpopulation, which is apparently a precursor of "classical" CD34+/133+ EPC, and functionally more potent than these with respect to homing and vascular repair.     

Expression of AC133 and CD117 on candidate normal stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia

To identify residual candidate normal progenitor/stem cell populations in childhood B-cell precursor acute lymphoblastic leukaemia (ALL), expression of AC133 and CD117 was analysed on the leukaemic cell clone and on immature B-lineage-negative CD34+CD19- bone marrow cells. 10/25 patients (40%) had no detectable expression of AC133 within the leukaemic cell clone. 24/26 patients (92%) lacked expression of CD117 on the leukaemic blast cell population. In contrast, a distinct AC133-positive cell population was found in 8/8 children with AC133-negative ALL and a CD117-positive cell population could be identified in 12/12 children with CD117-negative ALL, within the CD34+CD19- progenitor/stem cell compartment. These observations provide further evidence that in B-cell precursor ALL, unlike in acute myelogenous leukaemia, it may be possible to distinguish residual normal progenitor/stem cells from the leukaemic cell clone.     

Isolation and characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hematopoietic stem cells using cell surface markers AC133 and CD7

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34(-)Lin(-)). A major barrier in the further characterization of human CD34(-) stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34(-)CD38(-)Lin(-) and CD34(+)CD38(-)Lin(-) cells derived from human cord blood. Although the majority of CD34(-)CD38(-)Lin(-) cells lack AC133 and express CD7, an extremely rare population of AC133(+)CD7(-) cells was identified at a frequency of 0.2%. Surprisingly, these AC133(+)CD7(-) cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34(+) stem cells, and they were the only subset among the CD34(-)CD38(-)Lin(-) population capable of giving rise to CD34(+) cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo-cultured AC133(+)CD7(-) cells isolated from the CD34(-)CD38(-)Lin(-) population, whereas 400-fold greater numbers of the AC133(-)CD7(-) subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34(-) cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34(+) cells. (Blood. 2000;95:2813-2820)     

Immunophenotypic and functional characterization of CD33(+)CD34(+) cells in human cord blood of preterm neonates

OBJECTIVE: To characterize CD33(+)CD34(+) cells, a major population in human cord blood (CB) CD34(+) cells of preterm neonates. MATERIALS: The proportion of CD33(+) cells was analyzed on CB CD34(+) cells from preterm and full-term neonates. CD33(+)CD34(+) cells were purified by cell sorting and analyzed on their clonogenic activity, proliferative activity in short-time liquid suspension culture, and GATA-2 mRNA expression by RT-PCR and Southern blot. RESULTS: The absolute numbers and proportion of CD34(+) cells in mononuclear cells inversely correlated with gestational age. CD33 was expressed on a majority of CB CD34(+) cells of preterm neonates but on only a minor population of them in full-term neonates. In addition, CD33 was dominantly expressed on CD38(-)CD34(+) cells or CD117(low)CD34(+) cells in CB of preterm neonates. CD33(+)CD34(+) cells of preterm cord blood had high proliferative and reproducible potentials compared with CD33(-)CD34(+) cells. CD33(+)CD34(+) cells as well as CD33(-)CD34(+) cells from preterm CB highly expressed GATA-2, in contrast to those from BM. CONCLUSIONS: These results suggest that CD33(+)CD34(+) cells, which are a major population in CB CD34(+) cells of preterm neonates, do not simply represent relatively mature myeloid lineage hematopoietic progenitor cells as those in adult BM CD34(+) cells, and may contain hematopoietic stem cells or primitive progenitor cells as in fetal liver.     

Early haemoglobin-independent increase of plasma erythropoietin levels in patients with acute myocardial infarction.

AIMS: We studied plasma erythropoietin (EPO) levels and their relation with CD34(+)VEGFR-2(+) (mature and progenitor endothelial cells) and CD34(+) CD133(+)VEGFR-2(+), or CD34(+) CD117(+)VEGFR-2(+) (early/immature endothelial progenitors) cells in patients with acute myocardial infarction (AMI). METHODS AND RESULTS: Fifty AMI patients undergoing percutaneous coronary intervention (PCI) within 6 h of symptom onset were enrolled. EPO, measured by ELISA, and cell subsets, by cytofluorimetric analysis, were evaluated before PCI, 24 h and 7 days afterwards. Forty-five healthy subjects (CTRLs) were studied. Plasma EPO levels were higher in AMI patients at admission, 24 h, and 7 days (P = 0.04, P = 0.0001, P = 0.001, respectively) than in CTRLs. No correlation was evidenced between EPO and haemoglobin (Hb) or haematocrit at admission or 24 h after AMI. Differently, both Hb and haematocrit inversely correlated with EPO at day 7 (P = 0.0016, P = 0.029, respectively). Plasma EPO levels correlated with CD34(+)CD133(+)VEGFR-2(+) cells at day 7 (P = 0.03). CONCLUSION: AMI patients have increased plasma EPO levels until day 7. In the early phase, plasma EPO levels are Hb-independent; at day 7, an Hb-modulated increase of EPO correlates with the percentage of CD34(+)CD133(+)VEGFR-2(+) cells.     

Modulation of VEGFR-2-mediated endothelial-cell activity by VEGF-C/VEGFR-3

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of the VEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3(+) endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A- and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1-induced dispersion for maintenance of EC integrity.     

In vitro evidence for the presence of hematopoietic stem cells in the adult human liver

Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34(+) HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (+/-23%) of CD34(+) HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (+/-22%) of CD34(+) HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34(+) HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU-E), granulocyte-monocyte (CFU-GM), and mixed colonies (CFU-GEMM) were detected after culture of unseparated HMNCs and the enriched CD34(+) HMNC population; 14.3 +/- 13.2 (mean +/- SD) BFU-E, 3.1 +/- 3.1 CFU-GM, and 0.4 +/- 0.9 CFU-GEMM per 1 x 10(5) unseparated HMNCs and 16.0 +/- 9.5 BFU-E and 1.7 +/- 0.9 CFU-GM were identified per 2.4 x 10(3) CD34(+) HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation.     

Absence of a CD34- hematopoietic precursor population in recipients of CD34+ stem cell transplantation

The purified CD34(+) cell fraction has been used for hematopoietic stem cell transplantation since they were demonstrated to have long-term reconstituting ability. Therefore, the potential effects of CD34(-) stem cells on the clinical course have been a major concern in recipients of CD34(+)-selected transplantation. To address this concern, we used an in vitro assay to determine whether transplant recipients have CD34(-)precursor population. Lin(-)CD34(-) cells were isolated from bone marrow cells in 11 transplant recipients including four CD34-selected transplantations, six standard bone marrow transplantations, and one T cell-depleted marrow transplantation. The frequency of the Lin(-)CD34(-) population in four CD34-enriched transplantation recipients was not different from those of normal donors or recipients of other modes of transplantation: 0.96 +/- 1.01% (mean +/- s.d., n = 4), 0.45 +/- 0.16% (n = 6), and 0.66 +/- 0.59% (n = 7), respectively. However, the Lin(-)CD34(-)population obtained from the recipients of CD34-enriched transplantation acquired neither CD34 expression nor colony-forming activity after 7 days of culture, whereas the cells from all the normal individuals and standard BMT recipients were able to differentiate into CD34(+) cells accompanied by the emergence of colony-forming activity.We conclude that recipients of CD34-enriched transplantation appear to have defects in their CD34(-) precursor population. The clinical significance of these defects will be determined in a life-long follow-up of these patients.     

Presence of bone marrow-derived circulating progenitor endothelial cells in the newly formed lymphatic vessels

Bone marrow (BM)-derived circulating endothelial precursor cells (CEPCs) have been reported to incorporate into newly formed blood vessels under physiologic and pathologic conditions. However, it is unknown if CEPCs contribute to lymphangiogenesis. Here we show that in a corneal lymphangiogenesis model of irradiated mice reconstituted with enhanced green fluorescent protein (EGFP)-positive donor bone marrow cells, CEPCs are present in the newly formed lymphatic vessels. Depletion of bone marrow cells by irradiation remarkably suppressed lymphangiogenesis in corneas implanted with fibroblast growth factor-2 (FGF-2). Further, transplantation of isolated EGFP-positive/vascular endothelial growth factor receptor-3-positive (EGFP+/VEGFR-3+) or EGFP+/VEGFR-2+ cell populations resulted in incorporation of EGFP+ cells into the newly formed lymphatic vessels. EGFP+/CEPCs were also present in peritumoral lymphatic vessels of a fibrosarcoma. These data suggest that BM-derived CEPCs may play a role in "lymphvasculogenesis."     

Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells

Transplantation of ex vivo expanded human umbilical cord blood cells (hCB) only partially enhances the hematopoietic recovery after myelosuppressive therapy. Incubation of hCB with optimal combinations of cytokines and niche cells, such as endothelial cells (ECs), could augment the efficiency of hCB expansion. We have devised an approach to cultivate primary human ECs (hECs) in serum-free culture conditions. We demonstrate that coculture of CD34(+) hCB in direct cellular contact with hECs and minimal concentrations of thrombopoietin/Kit-ligand/Flt3-ligand resulted in a 400-fold expansion of total hematopoietic cells, 150-fold expansion of CD45(+)CD34(+) progenitor cells, and 23-fold expansion of CD45(+) Lin(-)CD34(hi+)CD45RA(-)CD49f(+) stem and progenitor cells over a 12-day period. Compared with cytokines alone, coculture of hCB with hECs permitted greater expansion of cells capable of multilineage engraftment and serial transplantation, hallmarks of long-term repopulating hematopoietic stem cells. Therefore, hECs establish a cellular platform for expansion of hematopoietic stem and progenitor cells and treatment of hematologic disorders.