[Show abstract][Hide abstract] ABSTRACT: Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.
[Show abstract][Hide abstract] ABSTRACT: The study was aimed to investigate the effect of deriving hematopoietic cells from human embryonic stem cells (hESCs) by the erythropoietin gene-modified conditioned medium of human mesenchymal cells. The mesenchymal stem cells (MSCs) steadily expressing EPO were established by lentiviral system. The expression of exogenous EPO was detected by RT-PCR and Western blot. After suspension culture, hESCs developed into embryonic bodies (EBs). Then the EB cells were cultured in conditional medium. The hESCs-derived hematopoietic cells were analyzed by immunofluorescence, CFU assay and RT-PCR. The results indicated that the exogenous EPO successfully expressed in the EPO transfected MSCs (EPO/MSCs). The supernatant from EPO/MSCs increased CD34(+) cell population and the expression of globin, and enhanced colony forming unit incidence. These effects were obviously higher than that of control. It is concluded that the EPO gene-modified conditioned medium of human mesenchymal cells can induce the hESCs to differentiate into hematopoietic cells.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 08/2010; 18(4):976-80.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have suggested that prostaglandin (PG) E2 (PGE2) and the prostaglandin pathway are essential for hematopoietic
stem cell growth and development. However, similar studies on hematopoietic commitment from human embryonic stem cells (hESCs)
are still limited. Here we report that the addition of PGE2 promotes hematopoietic differentiation of hESCs. The induced cells
from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by reverse transcription-PCR (RT-PCR), flow cytometry,
colony-forming assays and Wright-Giemsa staining. Our results demonstrated that PGE2 exposure could alter the gene expression
pattern and morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with higher frequencies
of CD34+ and CD45+ cells. Furthermore, the Smad signaling pathway may be involved in PGE2 and OP9 induced hematopoietic differentiation of hESCs.
This research may improve our knowledge of stem cell regulation and hopefully lead to better stem cell-based therapeutic options.
Keywordshuman embryonic stem cells-prostaglandin E2-hematopoiesis-
in vitro differentiation
[Show abstract][Hide abstract] ABSTRACT: To explore the mechanism of Flt3 receptor-interacting lectin (FRIL) maintains quiescence of hematopoietic stem cells (HSCs) in vitro.
Cord blood CD34+ cells were cultured in suspension medium supplemented with or without FRIL and FL. Cells were collected at different time points and the expression of some cell cycle regulators, especially those involved in G0/G1 phase regulation were detected on mRNA and protein level.
The expressions of G0/G1 phase related cyclins or CDKs were undetectable in the newly isolated CD34+ cells, expressions of Cyclin D3, CDK6 and P27 were the lowest in FRIL cultured group after 3d's culture (FRIL group: 483 +/- 63, 553 +/- 39, 0.312 +/- 0.030; FL group: 2437 +/- 52, 3209 +/- 98, 0.787 +/- 0.024; BLANK: 914 +/- 105, 1497 +/- 55, 0.616 +/- 0.029, respectively), but the expression of P53 was the highest in FRIL group (FRIL group: 4.476 +/- 0.159; FL group: 0.581 +/- 0.099, BLANK: 2.167 +/- 0.114). The expression of positive regulators of cell cycle in FRIL group were the same as that of FL group and blank group or lower.
FRIL preserves HSCs effectively in vitro through the mechanisms of down-regulation of cyclin D3 and CDK6 and activation of P53. P27 is mostly involved in the differentiation of HSCs.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 02/2007; 28(1):37-40.
[Show abstract][Hide abstract] ABSTRACT: Cell cycle progression is tightly regulated in hematopoietic stem cells. The cycle state decides cells' fates, which includes self-renewal, proliferation and differentiation. Proper cell cycle regulation is a pivotal element for the maintenance of hematopoiesis homeostasis. HTm4 is a newly identified specific cell cycle regulator of the hematopoietic cell. Through interacting with KAP-CDK2 complex, it arrests cells in G(0)/G(1) phase. K562 is a human chronic myelogenous leukemia cell; it could be induced to megakaryoblast by phorbol 12-myristate 13-acetate (PMA). Such differentiation must be associated with cell cycle change. To further clarify HTm4's function in hematopoietic cell cycle regulation, K562 cells were treated with PMA. Cell cycle change was analysed using flow cytometric system. And during the induction process gene expression of HTm4 as well as CycleE and CDK2, which are responsible for G(1) to S transition, were analysed using semi-quantitative RT-PCR. The C-terminal domain of HTm4 protein has been shown to be important for HTm4's binding with KAP-CDK2 complex. To determine its impact on HTm4's function, HTm4 and C-terminal truncated HTm4 (HTm4-ct) were transfected into K562 cells using Tet-Off regulation expression system. Their influence on cell cycle was observed. The results showed that PMA induced both expansion and differentiation of K562 cells as measured by cell number count and NBT staining respectively. During PMA treatment, G(0)/G(1) cell proportion and HTm4 expression displayed coordinated change, which suggested that HTm4 might drive K562 cells out of cell cycle but was not involved in the quiescence maintenance. Additionally, transfection of HTm4 caused G(0)/G(1) arrest in K562 cells, while transfection of HTm4-ct did not. It is therefore suggested that the C-terminal domain is important for the function of HTm4 in cell cycle regulation.
Sheng li xue bao: [Acta physiologica Sinica] 05/2005; 57(2):188-92.
[Show abstract][Hide abstract] ABSTRACT: Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.
Sheng li xue bao: [Acta physiologica Sinica] 07/2004; 56(3):306-12.