Umbilical Cord Blood-derived Stromal Cells Research Articles

Ginsenoside Rg1 protects human umbilical cord blood-derived stromal cells against tert-Butyl hydroperoxide-induced apoptosis through Akt-FoxO3a-Bim signaling pathway.

Human umbilical cord blood-derived stromal cells (hUCBDSCs) possess strong capability of supporting hematopoiesis and immune regulation, whereas some stress conditions cause reactive oxygen species (ROS) accumulation and then lead to oxidative injury and cell apoptosis. Ginsenoside Rg1 (G-Rg1) has been demonstrated to exert antioxidative and prosurvival effects in many cell types. In this study, the tert-Butyl hydroperoxide (t-BHP), an analog of hydroperoxide, was utilized to mimic the oxidative damage to hUCBDSCs. We aimed to investigate the effects of Ginsenoside Rg1 on protecting hUCBDSCs from t-BHP-induced oxidative injury and apoptosis, as well as the possible signaling pathway involved. It was shown that the treatment of hUCBDSCs with G-Rg1 markedly restored the t-BHP-induced cell viability loss, promoted the CFU-F formation, and inhibited cell apoptosis. G-Rg1 also caused a reduced production of LDH and MDA while significantly enhancing the activity of SOD. Mechanistically, G-Rg1 promoted the phosphorylation of Akt and FoxO3a and led to the cytoplasmic translocation of FoxO3a, which in turn suppressed FoxO3a-modulated expression of proapoptotic Bim and elevated the ratio of Bcl-2 to Bax. All these results suggest that G-Rg1 enhances the survival of t-BHP-induced hUCBDSCs and protects them against apoptosis at least partially through Akt-FoxO3a-Bim signaling pathway.

Induction of insulin-producing cells from umbilical cord blood-derived stromal cells by activation of the c-Met/HGF axis.

Efficient and effective therapies are required for diabetes mellitus. The use of adult stem cells for treating diabetes represents a major focus of current research. We have attempted to differentiate adult stem cells produced from umbilical cord blood-derived stromal cells into insulin-producing cells (IPCs). By activating the c-Met/HGF axis through temporal hypoxia treatment and hepatocyte growth factor (HGF) supplementation, our protocol resulted in the differentiation of cells into functional pancreatic endocrine cells with increased viability. Glucose stimulation test results showed that significantly greater amounts of C-peptide and insulin were released from the differentiated cells than from undifferentiated cells. These IPCs were capable of reversing the hyperglycemia of diabetic mice. In conclusion, targeting the c-Met/HGF axis can be considered an effective and efficient means of obtaining IPCs from adult stem cells.

Human umbilical cord blood-derived stromal cells: A new resource for the proliferation and apoptosis of myeloma cells

Background/ObjectiveBone marrow stromal cells (BMSCs) can support multiple myeloma (MM) disease progression and resistance to chemotherapy. The proliferation of MM cells may be suppressed by modifying the hematopoietic microenvironment (HME). We have previously isolated human umbilical cord blood-derived stromal cells (hUCBDSCs) and observed that hUCBDSCs suppressed proliferation and induced apoptosis in KM3 cells. To examine the mechanism by which hUCBDSCs drive the inhibition of MM, KM3 cells were co-cultured with hUCBDSCs.MethodsInterleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) expression levels were measured by enzyme-linked immunosorbent assay. The expression levels of membrane IL-6 receptor (mIL-6R), intercellular cell adhesion molecule-1 (ICAM-1), B-cell lymphoma/leukemia-2 (Bcl-2), and Bcl-XL as well as the location of nuclear factor κB (NF-κB) were assessed by laser confocal microscopy. The expression profiles of mIL-6R and ICAM-1 were also more precisely examined by flow cytometry, and Bcl-2, Bcl-XL and inhibitor kappa B expression levels were analyzed by western blot. The mRNA expression levels of IL-6R, ICAM-1, Bcl-2, and Bcl-XL were assessed by real-time polymerase chain reaction. NF-κB DNA-binding activity was examined by electrophoretic mobility shift assay.ResultsThe protein expression levels of both sIL-6R and mIL-6R were reduced in culture conditions when KM3 cells were co-cultured with hUCBDSCs; moreover, the mRNA expression levels of IL-6R were also reduced. Nuclear translocation of the NF-κB p65 subunit was inhibited in KM3 cells by co-culture with hUCBDSCs. Moreover, hUCBDSCs inhibited NF-κB DNA-binding activity, thereby resulting in the downregulation of NF-κB-regulated proteins.ConclusionhUCBDSCs can suppress proliferation and induce apoptosis in KM3 cells by both downregulating IL-6R expression and inhibiting NF-κB activity.

HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway

The human high-mobility group protein A2 (HMGA2) protein is an architectural transcription factor that transforms chromatin structure by binding to DNA. Recently, it has been reported that HMGA2 is highly expressed in fetal neural stem cells and has the capacity to promote stemness. However, there is currently no information available on the functional significance and molecular mechanisms of the cellular in vitro aging and proliferation of human umbilical cord blood-derived stromal cells (hUCBSCs). In the present study, we evaluated the direct effects of HMGA2 on the cellular aging and proliferation of hUCBSCs and investigated potential regulatory mechanisms responsible for the corresponding functions. We found that the overexpression of HMGA2 enhanced proliferation and reduced or even reversed the in vitro aging process of hUCBSCs. This effect was accompanied by the increased expression of cyclin E and CDC25A and the significantly decreased expression of cyclin-dependent kinase inhibitors. Furthermore, HMGA2 inhibition compromised cell proliferation and adipogenic differentiation in early-stage hUCBSCs. From the molecular/cellular functional analysis of microarray data, we found that HMGA2 overexpression induced a PI3K/Akt/mTOR/p70S6K cascade, which in turn suppressed the expression of p16INK4A and p21CIP1/WAF1 in hUCBSCs. These results provide novel insights into the mechanism by which HMGA2 regulates the in vitro aging and proliferation of hUCBSCs.

Umbilical Cord Blood-Derived Stromal Cells Regulate Megakaryocytic Proliferation and Migration Through SDF-1/PECAM-1 Pathway

We have previously reported that human umbilical cord blood-derived stromal cells (hUCBDSCs) are able to enhance the expansion of CFU-Meg in vitro, particularly promote the megakaryocytic lineage recovery, and effectively protect the survival of irradiated mice. In this study, we demonstrated that hUCBDSCs secreted SDF-1 to stimulate PECAM-1 expression in HEL cells (MK cell line), and consequently promoted the proliferation and migration of HEL cells. On the other hand, SDF-1 knock down in hUCBDSCs or PECAM-1 knock down in HEL cells diminished or abrogated the above effect. In addition, SDF-1/PECAM-1 probably activated PI3K/Akt and MAPK/ERK1/2 pathways. This report for the first time defines a SDF-1/PECAM-1 signaling pathway in the proliferation and migration of MKs, which provides supportive evidence for the clinical applications of hUCBDSCs in the treatment of megakaryocytic injury.

Hypothesis: Human Umbilical Cord Blood-Derived Stromal Cells Regulate the Foxp3 Expression of Regulatory T Cells Through the TGF-β1/Smad3 Pathway

Despite the improvements in transplant immunology and clinical and supportive care, graft-versus-host disease (GVHD) is still among the most common causes of overall mortality and morbidity after allogeneic hematopoietic cell transplantation. The development and severity of GVHD are strongly related with post-transplant outcomes. New strategies should be explored to overcome GVHD. Regulatory T cells (Treg cells), as dedicated suppressors of diverse immune responses and inflammation and important gatekeepers of immune homeostasis, contribute to the prevention of graft rejection and induce transplantation tolerance. Foxp3, a transcription factor, is predominantly expressed in Treg cells and is a master regulator of the development and function of Treg cells. Foxp3 mutations and Foxp3 deficiency lead to lethal autoimmune lymphoproliferative disease, which results from a defect in Treg cells. TGF-β1 is required to maintain Foxp3 expression in Treg cells. We isolated a novel population from among CD34(+) cells in our laboratory, referred to as human umbilical cord blood-derived stromal cells (hUCBDSCs), which exert an immunosuppressive effect and can notably increase Foxp3 expression in Treg cells. Our previous study also revealed that hUCBDSCs constantly secrete TGF-β1. Based on the literature searchings and our experimental findings, we hypothesize that hUCBDSCs, which secrete a high level of TGF-β1, modulate the Foxp3 expression of Treg cells through the TGF-β1/Smad3 pathway to regulate GVHD.

Human Umbilical Cord Blood-Derived Stromal Cells Are Superior to Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Inducing Myeloid Lineage Differentiation In Vitro

Stromal cells and mesenchymal stem cells (MSCs), 2 important cell populations within the hematopoietic microenvironment, may play an important role in the development of hematopoietic stem/progenitor cells. We have successfully cultured human umbilical cord blood-derived stromal cells (hUCBDSCs). It has been demonstrated that MSCs also exist in hUCB. However, we have not found any reports on the distinct characteristics of hUCBDSCs and human umbilical cord blood-derived mesenchymal stem cells (hUCBDMSCs). In this study, hUCBDSCs and hUCBDMSCs were isolated from the cord blood of full-term infants using the same density gradient centrifugation and cultured in the appropriate medium. Some biological characteristics and hematopoietic supportive functions were compared in vitro. hUCBDSCs were distinct from hUCBDMSCs in morphology, proliferation, cell cycle, passage, immunophenotype, and the capacity for classical tri-lineage differentiation. Finally, quantitative real-time polymerase chain reaction analysis revealed that granulocyte colony-stimulating factor (G-CSF) gene expression was higher in hUCBDSCs than that in hUCBDMSCs. Enzyme-linked immunosorbent assay revealed that the secretion of G-CSF, thrombopoietin (TPO), and granulocyte macrophage colony-stimulating factor (GM-CSF) by hUCBDSCs was higher than that by hUCBDMSCs. After coculture, the granulocyte/macrophage colony-forming units (CFU-GM) of hematopoietic cells from the hUCBDSC feeder layer was more than that from the hUCBDMSC feeder layer. Flow cytometry was used to detect CD34(+) hematopoietic stem/progenitor cell committed differentiation during 14 days of coculture; the results demonstrated that CD14 and CD33 expression in hUCBDSCs was significantly higher than their expression in hUCBDMSCs. This observation was also true for the granulocyte lineage marker, CD15. This marker was expressed beginning at day 7 in hUCBDSCs. It was expressed earlier and at a higher level in hUCBDSCs compared with hUCBDMSCs. In conclusion, hUCBDSCs are different from hUCBDMSCs. hUCBDSCs are superior to hUCBDMSCs in supporting hematopoiesis stem/progenitor cells differentiation into myeloid lineage cells at an early stage in vitro.

Human Umbilical Cord Blood-derived Stromal Cells, a New Resource in the Suppression of Acute Graft-versus-host Disease in Haploidentical Stem Cell Transplantation in Sublethally Irradiated Mice

Human umbilical cord blood-derived stromal cells (hUCBDSCs), a novel population isolated from CD34(+) cells by our laboratory, exerted an immunosuppressive effect on xenogenic T cells. This study aimed to investigate whether hUCBDSCs play a critical role in the suppression of acute graft-versus-host disease (aGVHD). The hUCBDSCs were co-cultured with splenocytes (SPCs) of donor C57BL/6 mice. The aGVHD in the recipient (B6×BALB/c) F1 mice was induced by the infusion of bone marrow cells and SPCs from donor mice following sublethal irradiation. The shift in vivo for hUCBDSCs was detected. The proliferation and cell cycle of SPCs were tested by cell counting kit-8 and flow cytometry, respectively. The expression of CD49b natural killer (NK) cells and CD3 T cells was detected by flow cytometry in co-culture and post-transplantation. IL-4, and IFN-γ were detected by ELISA in the serum of co-culture and post-transplantation. The survival time, body weight, clinical score, and histopathological score were recorded for mice post-transplantation. The hUCBDSCs promoted the proliferation of SPCs and significantly increased the ratio of the S and G(2)/M phase (p < 0.05). The hUCBDSCs significantly increased the expression of CD49b NK cells and IL-4 protein and decreased the expression of CD3 T cells and IFN-γ protein both in vitro and in vivo. The survival time of mice with co-transplantation of hUCBDSCs was significantly prolonged, and decreased clinical and histopathological scores were also observed. The hUCBDSCs were continually detected in the target organs of GVHD. These results suggest that hUCBDSCs possess the capability of suppressing aGVHD, possibly via their influence on CD3 T cells, NK cells, and cytokines.

Cotransplantation of human umbilical cord blood-derived stromal cells enhances hematopoietic reconstitution and engraftment in irradiated BABL/c mice

Our previous study demonstrated that human umbilical cord blood-derived stromal cells (hUCBDSCs) support the growth of hematopoietic stem cells, and promote the expansion of colony-forming units of megakaryocyte (CFU-Mk) even more efficiently than human bone marrow stromal cells (hBMSCs). Given the unique role of hUCBDSCs in megakaryocytopoiesis in vitro, in this follow up study, we further investigated their effects in vivo on hematopoiesis in the setting of hematopoietic stem cell (HSC) transplants. After infusing hematopoietic cells alone or together with either hUCBDSCs or hBMSCs into lethally irradiated BABL/c mice, we examined the engraftment of human CD45+ cells in the mouse bone marrow with flow cytometry, observed the survival rate of irradiated mice, obtained blood counts and bone marrow biopsies at different time points post-transplant, and assessed colony forming and the homing efficiency of hematopoietic cells. By comparing HSC transplantation with or without stromal cells, we show here that hUCBDSCs efficiently promote the homing of hematopoietic cells to the marrow and enhance the engraftment after cotransplantation. They also promote hematopoietic reconstitution, particularly of the megakaryocytic lineage, and restore the impaired stromal microenvironment after radiation-induced damages. These effects of hUCBDSCs are more potent than those of hBMSCs. In conclusion, our findings suggest the role of hUCBDSCs to facilitate hematopoietic reconstitution and engraftment when cotransplanted with hematopoietic cells.

Human umbilical cord blood-derived stromal cells prevent graft-versus-host disease in mice following haplo-identical stem cell transplantation

Human umbilical cord blood-derived stromal cells (hUCBDSC) comprise a novel population of CD34(+) cells that has been isolated in our laboratory. They have been shown previously not only to be non-immunogenic but also to exert immunosuppressive effects on xenogenic T cells in vitro. This study investigated the role of hUCBDSC in immunomodulation in an acute graft-versus-host disease (GvHD) mouse model after haplo-identical stem cell transplantation. Acute GvHD was induced in recipient (B6 × BALB/c)F(1) mice by irradiation (750 cGy) followed by infusion of bone marrow cells and splenocytes from donor C57BL/6 mice. hUCBDSC were co-transplanted in the experimental group. The survival time, body weight and clinical and histopathologic scores were recorded after transplantation. The expression of surface markers [major histocompatibility complex (MHC) I, MHC II, CD80 and CD86] on CD11c(+) dendritic cells (DC), and the percentage of CD4(+) regulatory T cells (Treg), in the spleens of recipient mice were examined by flow cytometry. The survival time was significantly prolonged, and the clinical and histopathologic scores were reduced in mice co-transplanted with hUCBDSC. The expression levels of the surface markers on DC were significantly lower in mice transplanted with hUCBDSC compared with those without. The proportion of CD4(+) Treg in the spleen was also increased in mice transplanted with hUCBDSC. These results from a GvHD mouse model are in agreement with previous in vitro findings, suggesting that hUCBDSC possess immunosuppressive properties and may act via influencing DC and CD4(+) Treg.

Osteogenic differentiation of equine cord blood multipotent mesenchymal stromal cells within coralline hydroxyapatite scaffolds in vitro

To investigate the osteogenic differentiation potential of equine umbilical cord blood-derived multipotent mesenchymal stromal cells (CB-MSC) within coralline hydroxyapatite scaffolds cultured in osteogenic induction culture medium. Scaffolds seeded with equine CB-MSC were cultured in cell expansion culture medium (control) or osteogenic induction medium (treatment). Cell viability and distribution were confirmed by the MTT cell viability assay and DAPI nuclear fluorescence staining, respectively. Osteogenic differentiation was evaluated after 10 days using reverse transcription polymerase chain reaction, alkaline phosphatase activity, and secreted osteocalcin concentration. Cell morphology and matrix deposition were assessed by scanning electron microscopy (SEM) after 14 days in culture. Cells showed viability and adequate distribution within the scaffold. Successful osteogenic differentiation within the scaffolds was demonstrated by the increased expression of osteogenic markers such as Runx2, osteopontin, osteonectin, collagen IA; increased levels of alkaline phosphatase activity; increased osteocalcin protein secretion and bone-like matrix presence in the scaffold pores upon SEM evaluation. These results demonstrate that equine CB-MSC maintain viability and exhibit osteogenic potential in coralline hydroxyapatite scaffolds when induced in vitro . Equine CB-MSC scaffold constructs deserve further investigation for their potential role as biologically active fillers to enhance bone-gap repair in the horse.

Effects of Bone Marrow Stromal Cells and Umbilical Cord Blood-Derived Stromal Cells on Daunorubicin-Resistant Residual Jurkat Cells

Objective To observe the effects of the hematopoietic inductive microenvironment (HIM) simulated by stromal cells of different origins on daunorubicin-resistant residual Jurkat cells (Jurkat/DNR cells). Methods Jurkat/DNR cells were cultured and identified. Human umbilical cord blood-derived stromal cells (UCBDSCs) and normal human bone marrow stromal cells (BMSCs) were isolated and cocultured with Jurkat/DNR cells. Jurkat/DNR cells were collected after 14 days of coculture and analyzed with regard to cell proliferation and differentiation abilities, apoptosis, drug sensitivity, and MRD1 multidrug resistance gene mRNA expression. Results UCBDSC-simulated HIM suppressed proliferation and promoted apoptosis, differentiation, and drug sensitivity of Jurkat/DNR cells more significantly than BMSC-simulated HIM. Conclusions Both BMSCs and UCBDSCs reconstruct the leukemic HIM and reverse drug resistance in Jurkat/DNR cells. UCBDSCs reconstruct the leukemic HIM and reverse drug resistance more significantly than BMSCs.

Hypothesis: Human Umbilical Cord Blood-derived Stromal Cells Promote Megakaryocytopoiesis Through the Influence of SDF-1 and PECAM-1

The development and maturation of megakaryocytes (MKs) is a complex and multistage cellular and biological process. The hematopoietic microenvironment plays an important role in megakaryocytopoiesis regulation. Stromal cells, an important ingredient in the hematopoietic microenvironment, may regulate the development of MKs via the adhesion with MKs and via augmentation of cytokine secretion. Our laboratory has previously isolated a novel population of stromal cells from human umbilical cord blood, called hUCBDSCs. Compared with hBMSCs, the hUCBDSCs express higher levels of stromal cell-derived factor-1 (SDF-1) and increase the colony-forming-unit-megakaryocytes (CFU-MK). Meanwhile, there are reports identified a migration defect in PECAM-1-deficient MKs in response to a gradient of SDF-1. Based on literature searches and our experimental findings, we present a hypothesis that hUCBDSCs, secreting high level of SDF-1, modulated the expression of PECAM-1 of MKs, to regulate the megakaryocyte development.

Differentiation of cryopreserved human umbilical cord blood-derived stromal cells into cells with an oligodendrocyte phenotype

In this study, we examined the phenotypic characteristics of human umbilical cord blood-derived mesenchymal stromal cells (UCB-derived MSCs) differentiated along an oligodendrocyte pathway. We induced human UCB-derived MSCs to form floating neurospheres, and these neurospheres were then induced to differentiate into oligodendrocyte progenitor-like cells using multiple induction factors. Differentiated UCB-derived MSCs showed morphologic characteristics of an oligodendrocyte phenotype. The expression of cell surface markers characteristic of oligodendrocyte progenitor cells or oligodendrocytes was determined by immunocytochemical staining. These results suggest that human UCB-derived MSCs can be induced to differentiate into cells with an oligodendrocyte phenotype and that these cells may have potential in the future cellular therapy of central neurological disorders.

Human umbilical cord blood-derived stromal cells: Multifaceted regulators of megakaryocytopoiesis

It has been demonstrated that stromal cell precursors exist in human umbilical cord blood. After being cultured in vitro, these cells are called human umbilical cord blood-derived stromal cells (hUCBDSCs). However, the role of hUCBDSCs in hematopoiesis is still unclear. We have previously shown that hUCBDSCs are superior to human bone marrow stromal cells (hBMSCs) at enhancing the expansion of megakaryocyte colony forming units (CFU-Meg). Based on this observation, we postulated that hUCBDSCs might promote megakaryocytopoiesis. To test this hypothesis, we developed a megakaryocyte/hUCBDSC co-culture model and a hematopoietic microenvironment injury model in nude mice. We explored the ability and mechanisms by which hUCBDSCs promoted the proliferation of megakaryocytes in vitro, and we also explored their capacity to restore the hematopoietic microenvironment in vivo. As expected, hUCBDSCs were more effective than hBMSCs at enhancing the proliferation of megakaryocyte lines from HEL cells and restoring megakaryocytopoiesis in a hematopoietic microenvironment injury model in nude mice. Thrombopoietin (TPO) and stromal cell derived factor-1 (SDF-1) are two of the key factors underlying this capacity. We also found that gap junction intercellular communication (GJIC) between HEL cells and hUCBDSCs might be partially absent. Our data provide the first evidence that hUCBDSCs play a regulatory role during megakaryocytopoiesis, which might be important for designing treatments for patients with megakaryocytic injury.

Human Umbilical Cord Blood-derived Stromal Cells Suppress Xenogeneic Immune Cell Response In Vitro

To explore immunological properties of human umbilical cord blood-derived stromal cells (hUCBDSC) and their effect on xenogeneic immune cells in vitro. Immunological phenotype of freshly isolated and cryopreserved hUCBDSCs was evaluated by flow cytometry. Xenogeneic splenic T-cells were stimulated by phytohemaglutinin A (PHA) or dendritic cells in the absence or presence of hUCBDSCs. T-cell proliferation was measured by cell counting kit-8 after 7-day incubation. The proportion of apoptotic cells and CD4+CD25+Foxp3+ regulatory T-cells (Tregs) was determined in T-cells activated by PHA in the absence or presence of hUCBDSCs by flow cytometry. Phenotype of dendritic cells, cultured alone or with hUCBDSCs, was analyzed by flow cytometry. Levels of immune molecule expression on freshly isolated hUCBDSCs were as follows: human leukocyte antigen-I (HLA-I) (84.1+/-2.9%), HLA-II (1.6+/-0.3%), CD80 (0.8+/-0.1%), CD86 (0.8+/-0.1%), CD40 (0.6+/-0.1%), and CD40L (0.5+/-0.1%), which was not influenced by cryopreservation. T-cell proliferation in the presence of hUCBDSCs was significantly lower than that of positive control. The coculture led to a 10-fold increase (from 1.2+/-0.3% to 12.1+/-1.4%, P<0.001) in the proportion of CD4+CD25+Foxp3+ regulatory T-cells (Tregs) and a reversion of mature dendritic cells, as indicated by the down-regulation of major histocompatibility complex (MHC)-II molecule (49.3% vs 25.9%, P=0.001), CD80 (47.2% vs 23.3%, P=0.001), and CD86 (40.6% vs 25.1%, P=0.002). When subjected to annexin V binding and propidium iodide uptake assay, the hUCBDSCs did not show the ability to induce apoptosis of xenogeneic T-cells. These results demonstrate low immunogenicity and immunomodulation effect of the hUCBDSCs. Reversion of mature dendritic cells and increase in Treg proportion, but not cell apoptosis, can possibly contribute to the suppression of xenogeneic T-cell proliferation by the hUCBDSCs.

Human umbilical cord blood-derived stromal cell, a new resource of feeder layer to expand human umbilical cord blood CD34 + cells in vitro

Allogeneic transplantation with human umbilical cord blood (hUCB) in adult recipients is mainly limited by a low CD34 + cell dose. To break the limit, hUCB as a novel source of hUCB-derived stromal cells was incorporated in an attempt to expand CD34 + cells from hUCB in vitro. Cord blood CD34 + cells were separated by MACS system. HUCB-derived stromal cells were cultured by the Dexter system and characterized by morphologic, immunophenotypical, and functional analysis. We studied the effects of hUCB-derived stromal cells, cytokines, and hUCB-derived stromal cells combined with cytokines on expansion of hUCB CD34 + cells. The CD34 + cells were assessed for the degree of expansion and the number of colony-forming units in semisolid culture. Our research found that hUCB-derived stromal cells were mainly composed of three kinds of cell components, with CD106, CD29, CD44, CD45, CD50, CD68, CD31, Fn, Lm, and collagen IV positive, but CD34 negative immunophenotype. Functionally, it was discovered by cell cycle and growth curve analyses that the capability of colony and parietal layer formation of hUCB-derived stromal cells was poorer than that of BM stromal cells, and the doubling time of hUCB-derived stromal cells was longer than that of BM stromal cells. It was indicated by ELISA and RT-PCR that hUCB-derived stromal cells express higher level of TPO and less GM-CSF and SCF than BM stromal cell. Adherent layer of hUCB-derived stromal cells alone or combining with cytokines, increased CD34 + cell expansion. In vitro formation of CFUs by expanded CD34 + cells was significantly higher than that of unexpanded CD34 + cells ( P < 0.05). When cocultured with hUCB-derived stromal cells in the presence of cytokines, cell growth was significantly enhanced: CD34 + cells by 8.02 ± 0.96-fold, CFU-GM by 217.60 ± 6.72-fold, CFU-E by 1940.80 ± 52.78-fold, and CFU-Mg by 142.60 ± 4.39-fold. HUCB-derived stromal cells have significant superiority on the expansion of CFU-Mg ( P < 0.05). The results indicate that human umbilical cord blood-derived stromal cells may be a suitable feeder layer for expansion of hematopoietic progenitors from hUCB in vitro.