Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4

The periodontal ligament (PDL) comprises adult stem cells, which are responsible for periodontal tissue regeneration. In the present study, we investigated the specific profile of the stem cells in the human PDL. Microscopic analysis demonstrated that PDL cells showed a fibroblastic appearance, forming flat and loose aggregates. PDL cells expressed embryonic stem cell-associated antigens (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, NANOG, SOX2, and REX1, and alkaline phosphatase activity), as well as conventional mesenchymal stem cell markers. When PDL cells were cultured in the presence of all-trans-retinoic acid, the numbers of SSEA-3+ and SSEA-4+ PDL cells were significantly decreased, while that of SSEA-1+ was increased. SSEA-4+ PDL cells showed a greater telomere length and growth rate. SSEA-4+ PDL cells exhibited the potential to generate specialized cells derived from three embryonic germ layers: mesodermal (adipocytes, osteoblasts, and chondrocytes), ectodermal (neurons), and endodermal (hepatocytes) lineages. Our findings demonstrated that SSEA-4, a major antigen to distinguish human embryonic stem cells, could also be used to identify multipotent stem cells in the PDL. Hence, SSEA-4+ human PDL cells appear to be a promising source of stem cells for regenerative medicine.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

PDL1 Expression on Plasma and Dendritic Cells in Myeloma Bone Marrow Suggests Benefit of Targeted anti PD1-PDL1 Therapy

In this study we set out to investigate whether anti PDL1 or PD–1 treatment targeting the immune system could be used against multiple myeloma. DCs are important in regulating T cell responses against tumors. We therefore determined PDL1 and PDL2 expression on DC populations in bone marrow of patients with plasma cell disorders using multicolour Flow Cytometry. We specifically looked at CD141⁺ and CD141^- myeloid and CD303⁺ plasmacytoid DC. The majority of plasma cells (PC) and DC subpopulations expressed PDL1, but the proportion of positive PDL1+ cells varied among patients. A correlation between the proportion of PDL1⁺ PC and CD141⁺ mDC was found, suggesting both cell types could down-regulate the anti-tumor T cell response.     

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients.MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database.ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome.ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.     

An MRI-Visible Non-Viral Vector Bearing GD2 Single Chain Antibody for Targeted Gene Delivery to Human Bone Marrow Mesenchymal Stem Cells

The neural ganglioside GD2 has recently been reported to be a novel surface marker that is only expressed on human bone marrow mesenchymal stem cells within normal marrow. In this study, an MRI-visible, targeted, non-viral vector for effective gene delivery to human bone marrow mesenchymal stem cells was first synthesized by attaching a targeting ligand, the GD2 single chain antibody (scAb_GD2), to the distal ends of PEG-g-PEI-SPION. The targeted vector was then used to condense plasmid DNA to form nanoparticles showing stable small size, low cytotoxicity, and good biocompatibility. Based on a reporter gene assay, the transfection efficiency of targeting complex reached the highest value at 59.6% ± 4.5% in human bone marrow mesenchymal stem cells, which was higher than those obtained using nontargeting complex and lipofectamine/pDNA (17.7% ± 2.9% and 34.9% ± 3.6%, respectively) (P<0.01). Consequently, compared with the nontargeting group, more in vivo gene expression was observed in the fibrotic rat livers of the targeting group. Furthermore, the targeting capacity of scAb_GD2-PEG-g-PEI-SPION was successfully verified in vitro by confocal laser scanning microscopy, Prussian blue staining, and magnetic resonance imaging. Our results indicate that scAb_GD2-PEG-g-PEI-SPION is a promising MRI-visible non-viral vector for targeted gene delivery to human bone marrow mesenchymal stem cells.     

Placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells support hematopoietic recovery of X-irradiated human CD34+ cells

AimsThe potential of human mesenchymal stem cell-like stroma prepared from placental/umbilical cord blood for hematopoietic regeneration by X-irradiated hematopoietic stem cells is herein assessed.Main methodsPlacental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells were applied to a regenerative ex vivo expansion of X-irradiated human CD34⁺ cells in a serum-free liquid culture supplemented with a combination of interleukine-3 plus stem cell factor plus thrombopoietin.Key findingsThe total number of cells and of lineage-committed myeloid hematopoietic progenitor cells generated in the co-culture of both non-irradiated and X-irradiated cells with stromal cells was significantly higher than those in the stroma-free culture. In addition, the number of CD34⁺ cells and CD34⁺/CD38^? cells, immature hematopoietic stem/progenitor cells also increased more than the stroma-free culture. The stromal cells produced various types of cytokines, although there was little difference between the co-cultures of non-irradiated and X-irradiated cells with stromal cells. Furthermore, when X-irradiated cells came in contact with stromal cells for 16 h before cytokine stimulation, a similar degree of hematopoiesis was observed, thus suggesting the critical role of cell-to-cell interaction.SignificanceThe present results showed the potential efficacy of human mesenchymal stem cell-like stroma for hematopoietic regeneration from irradiated hematopoietic stem/progenitor cells.     

CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization andpurification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton’s jelly among others.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

In vitro Proliferation of Human Bone Marrow Mesenchymal Stem Cells Employing Donor Serum and Basic Fibroblast Growth Factor

A method for the in vitro proliferation of human bone marrow mesenchymal stem cells (MSCs) employing a medium not containing fetal calf serum (FCS) was developed for a regenerative medicine of cartilage using MSCs. Without using density-gradient centrifugation, the bone marrow aspirate was poured into a dish (6.0 \times 10⁵ nucleated cells/cm²) with DMEM medium containing 10% serum (FCS or donor serum) and basic fibroblast growth factor, and incubated at 37 °C under a 5% CO₂ atmosphere. The density of adhesive cells incubated with the medium containing human serum and basic fibroblast growth factor (10 ng/ml) almost reached confluence at 19d and was 1.4-2.7 times that in the medium containing only FCS. The density of cells incubated with the medium containing only human serum was 0.1-0.6 times that in the medium containing only FCS. The content of CD45^- CD105⁺ cells among the cells harvested after a 19-d incubation in the medium containing human serum and basic fibroblast growth factor was higher than 90%. This high content and chondrogenic activity, which was confirmed by pellet cultivation and staining with Safranine O, were maintained even after further subcultivation in the medium to 17 population doubling levels. Consequently, this method might be applicable to in vitro proliferation of MSCs for the regeneration of cartilage.     

Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34⁺ hematopoietic cells. Co-transplantation of CD34⁺ HSCs and CD34⁻ MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34⁺ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34⁺ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34⁺ cells. Based on additional in vitro results of endothelial differentiation of CD34⁺ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34⁺ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34⁺ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.     

Immunophenotype and differentiation capacity of bone marrow-derived mesenchymal stem cells from CBA/Ca,ICR and Balb/c mice

AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented Dulbecco’s modified Eagle’s medium.MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106,Sca-1 and CD44 and negativity to CD45,CD11b and MHC classⅡ.Differentiation capacity of MSC towards osteogenic and adipo-genic lineages were also assessed. RESULTS:MSC were successfully cultured from bone marrow of all 3 strains,albeit differences in the temporal expression of certain surface antigens.Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype(CD106-CD45+CD11b+Sca-1low)to typical MSC phenotype(CD106+CD45-CD11b-Sca-1high)with increasing passages. CONCLUSION:Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.     

Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1’s telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1^L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1^L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1^L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1^L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.     

Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow

Omentum fat derived stem cells have emerged as an alternative and accessible therapeutic tool in recent years in contrast to the existing persuasive sources of stem cells, bone marrow and subcutaneous adipose tissue. However, there has been a scanty citation on human omentum fat derived stem cells. Furthermore, identification of specific cell surface markers among aforesaid sources is still controversial. In lieu of this existing perplexity, the current research work aims at signifying omentum fat as a ground-breaking source of stem cells by surface antigenic profiling of stem cell population. In this study, we examined and compared the profiling of cell surface antigenic expressions of hematopoietic stem cells, mesenchymal stem cells, cell adhesion molecules and other unique markers such as ABCG2, ALDH and CD 117 in whole cell population of human omentum fat, subcutaneous fat and bone marrow. The phenotypic characterization through flowcytometry revealed the positive expressions of CD 34, CD 45, CD 133, HLADR, CD 90, CD 105, CD 73, CD 29, CD 13, CD 44, CD 54, CD 31, ALDH and CD 117 in all sources. The similarities between the phenotypic expressions of omentum fat derived stem cells to that of subcutaneous fat and bone marrow substantiates that identification of ultimate source for curative therapeutics is arduous to assess. Nevertheless, these results support the potential therapeutic application of omentum fat derived stem cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9427-4) contains supplementary material, which is available to authorized users.     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.     

Hematopoietic stem cell expansion caused by a synthetic fragment of leptin

Leptin is a cytokine that regulates food intake, energy expenditure and hematopoiesis. Based on the tridimensional structure of the human leptin molecule, six fragments have been synthesized, (Ac-Lep_23–47-NH₂, [LEP1]; Ac-Lep_48–71-NH₂, [LEP2]; Ac-Lep_72–88-NH₂, [LEP3]; Ac-Lep_92–115-NH₂, [LEP4], Ac-[Ser¹¹⁷]-Lep_116–140-NH₂, [LEP5] and Ac-Lep_141–164-NH₂, [LEP6]), and their effects on hematopoiesis were evaluated. The mice were treated with 1 mg/kg LEP5 for 3 days. The mature and primitive hematopoietic populations were quantified. We observed that the mature populations from the bone marrow and spleen were not affected by LEP5. However, the peptide caused at least a two-fold increase in the number of hematopoietic stem cells, the most primitive population of the bone marrow. Additionally, the number of granulocyte/macrophage colony-forming units produced by bone marrow cells in methylcellulose also increased by 40% after treatment with LEP5, and the leptin receptor was activated. These results show that the leptin fragment LEP5 is a positive modulator of the in vivo expansion of hematopoietic stem cells.     

Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b^−/− mTR^+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.     

Back Cover Image,Volume 117, Number 1, January 2020

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264⁺ hBM-MSCs from two age-matched donors have elevated β-galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264⁻ hBM-MSCs. Counterintuitive to their aging phenotype, CD264⁺ hBM-MSCs exhibited comparable survival to matched CD264⁻ hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264⁺ cells in hBM-MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.