The comparison of different protocols for expansion of umbilical-cord blood hematopoietic stem cells

Hematopoiesis is maintained by the activity of multipotent stem cells, which have the dual capacity to self-renew and to differentiate into all of the blood cell lineages. The major challenge of stem cells based regenerative therapy is to expand ex vivo the primitive compartment to increase transplantable stem cells number. The present study was designed to evaluate several culture systems for in vitro maintenance of umbilical cord blood stem cells. The influences of different growth conditions such as stromal feeder layer, cytokines supplement and placental conditioned medium (PCM) have been evaluated over a relatively short period of time on CD34(+) cell expansion and maintenance of clonogenic progenitors. When cells were expanded on feeder layer in the presence of added cytokines and PCM on average a 2.96-fold increase of CD34(+)CD71(-) and a 3.13-fold increase of CD34(+)HLA-DR(-) was observed. The total number of colony forming cells (35 +/- 2.65) indicated also that the yield of clonogenic progenitors obtained with a combination of all factors was two folds higher than each of these factors alone and ten time above control (3.67 +/- 2.52). In conclusion, the results of our study clearly show that the ex vivo expansion of hematopoietic progenitor cells obtained from human umbilical cord blood is dependent on controlled experimental conditions, which might be helpful when designing culture systems for clinical applications.     

Transplantation of hematopoietic stem cells from the peripheral blood

Hematopoietic stem cells can be collected from the peripheral blood. These hematopoietic stem cells (HSC), or better progenitor cells, are mostly expressed as the percentage of cells than react with CD34 antibodies or that form colonies in semi-solid medium (CFU-GM). Under steady-state conditions the number of HSC is much lower in peripheral blood than in bone marrow. Mobilization with chemotherapy and/or growth factors may lead to a concentration of HSC in the peripheral blood that equals or exceeds the concentration in bone marrow. Transplantation of HSC from the peripheral blood results in faster hematologic recovery than HSC from bone marrow. This decreases the risk of infection and the need for blood-product support. For autologous stem-cell transplantation (SCT), the use of peripheral blood cells has completely replaced the use of bone marrow. For allogeneic SCT, on the other hand, the situation is more complex. Since peripheral blood contains more T-lymphocytes than bone marow, the use of HSC from the peripheral blood increases the risk of graft-versus-host disease after allogeneic SCT. For patients with goodrisk leukemia, bone marrow is still preferred, but for patients with high-risk disease, peripheral blood SCT has become the therapy of choice.     

Tolerance induction by allogeneic hematopoietic stem cells

In this issue of Cell Stem Cell, Zheng et?al. (2011) report that HSCs expressing PD-L1 display enhanced engraftment in irradiated allogeneic recipients. Independently in Nature, Fujisaki et?al. (2011) observe allogeneic HSCs persisting in proximity to regulatory T?cells in nonirradiated recipients, further connecting HSCs and immune tolerance.     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents

BMSCs (bone‐marrow‐derived mesenchymal stem cells) and ADSCs (adipose tissue‐derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis‐related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose‐dependent reduction in cell viability compared with 5‐FU (5‐fluorouracil) treatment. Different expression patterns of apoptosis‐related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy‐induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.     

Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth suplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65 ± 0.2% and CD106/VCAM 7.73±0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.     

Derivation of hematopoietic stem cells from murine embryonic stem cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny. Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability. Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types. The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol. HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders. However, many patients that might benefit from HSC therapy lack access to suitable donors. ESC could provide an alternative source of HSC for these patients. The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC. In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation. EBs are then dissociated and infected with retroviral HoxB4. Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days. During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100 s of millions of cells. These cells can then be used to rescue and reconstitute lethally irradiated mice.     

Optimization of cryopreservation condition for hematopoietic stem cells from umbilical cord blood

The conditions for cryopreservation of CD34⁺ hematopoietic stem cells (HSC) from umbilical cord blood (UCB) were optimized with a new cryo-medium containing 10% ethylene glycol (EG) and 2% dimethyl sulfoxide (Me₂SO) using a controlled-rate freezing (CRF) method. After the cryopreservation of mononuclear cells (MNC) from UCB, recoveries of MNC, CD34⁺ cells, and total colony-forming units (CFU) were significantly improved compared to those in the control cryo-medium containing 10% Me₂SO and 2% Dextran-40 (P < 0.05). This study shows that the new cryo-medium and CRF method provide better recoveries of MNC, HSC and total CFU than the control cryo-medium and isopropylalcohol freezing (IPA) method. Therefore, this cryo-medium, combined with the CRF method, is valuable for optimizing cryopreservation conditions for HSC from UCB to obtain satisfactory HSC recovery.     

UC blood hematopoietic stem cells and therapeutic angiogenesis

Studies suggest that mobilized hematopoietic stem cells (HSC) are recruited to ischemic tissue and stimulate angiogenesis. Critical observations in pre-clinical studies have identified an augmentation of endogenous microvascular collateralization that is beyond that directly attributable to anatomic incorporation and differentiation of infused human cells into the vascular endothelium. Evidence links age-associated reductions in the levels of circulating marrow-derived HSC characterized by expression of early HSC markers CD133 and CD34, with the occurrence of cardiovascular events and associated death. Utilizing the patient's own HSC to augment angiogenesis has several disadvantages, including reduced function of these cells and logistical issues related to cell collection from individual patients. Thus an available source of allogeneic HSC such as UC blood for cellular therapy may be optimal.     

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34(+) stem cells (up to 1.95 x 10(6)-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.     

Hepatic regeneration from hematopoietic stem cells

In recent years, numerous investigators have reported novel cellular fates of multipotent stem or progenitor cells. In this review, we discuss the unexpected observations that hematopoietic stem cells can contribute to the hepatocyte lineage in humans and in rodent models of liver disease and regeneration. A key unresolved issue regarding hepatic regeneration from hematopoietic stem cells is whether the mechanism occurs through transdetermination, cell fusion, or other processes. A better understanding of the various stem or progenitor cells of the hepatic lineage may facilitate cellular transplantation approaches for the correction of hepatic function in patients with end-stage liver disease.     

Characteristics of hematopoietic stem cells of umbilical cord blood

Umbilical cord blood collected from the postpartum placenta and cord is a rich source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow transplantation. In this review we wanted to describe the differences (in phenotype, cytokine production, quantity and quality of cells) between stem cells from umbilical cord blood, bone marrow and peripheral blood. HSCs present in cord blood are more primitive than their counterparts in bone marrow or peripheral blood, and have several advantages including high proliferation. With using proper cytokine combination, HSCs can be effectively developed into different cell lines. This process is used in medicine, especially in hematology.     

Lymphocyte development from hematopoietic stem cells

The recent application of new techniques, such as multi-color cell sorting and the production of transgenic and gene-knockout mice, has contributed to a better understanding of lymphocyte development from hematopoietic stem cells. Now that we can purify progenitors at different maturational stages during lymphocyte development, the challenge is to understand the processes that govern each developmental stage transition.     

Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood

BACKGROUND: Umbilical cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSC/HPC) for the reconstitution of the hematopoietic system after clinical transplantation. Cryopreservation of these cells is critical for UCB banking and transplantation as well as for research applications by providing readily available specimens. The objective of this study was to optimize cryopreservation conditions for CD34+ HSC/HPC from UCB. METHODS: Cryopreservation of CD34+ HSC/HPC from UCB after mononuclear cell (MNC) preparation was tested in a research-scale setup. Experimental variations were concentration of the cryoprotectant, the protein additive and cell concentration. In addition, protocols involving slow, serial addition and removal of DMSO were compared with standard protocols (fast addition and removal of DMSO) in order to avoid osmotic stress for the cryopreserved cells. Viability and recoveries of MNC, CD34+ cells and total colony-forming units (CFU) were calculated as read-outs. In addition, sterility testing of the collected UCB units before further processing was performed. RESULTS: The optimal conditions for cryopreservation of CD34+ HPC in MNC preparations were 10% DMSO and 2% human albumin at high cell concentrations (5 x 10(7) MNC/mL) with fast addition and removal of DMSO. After cryopreservation using a computer-controlled freezer, high viabilities (89%) and recoveries for CD34+ cells (89%) as well as for CFU (88%) were observed. Microbial contamination of the collected UCB samples was reduced to a rate of 6.4%. DISCUSSION: Optimized cryopreservation conditions were developed for UCB MNC in respect of the composition of the cryosolution. In addition, our results showed that fast addition of DMSO is essential for improved cryopreservation and post-thaw quality assessment results, whereas the speed of DMSO removal after thawing has little influence on the recoveries of CD34+ cells and CFU.     

Expansion of human cord blood hematopoietic stem cells for transplantation

A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.     

The immunologic and hematopoietic profiles of mesenchymal stem cells derived from different sections of human umbilical cord

Mesenchymal stem cells （MSCs） have been widely used in allogeneic stem cell transplantation. We compared im- munologic and hematopoietic characteristics of MSCs derived from whole human umbilical cord （UC）, as well as from different sections of UCs, including the amniotic membrane （AM）, Wharton＇s jelly （WJ）, and umbilical vessel （UV）. Cell phenotypes were examined by flow cytometry. Lymphocyte transformation test and mixed lymphocyte reaction were performed to evaluate the immuno-modulatory activity of MSCs derived from UCs. The mRNA expression of cytokines was detected by real- time polymerase chain reaction. Hematopoietic function was studied by co-culturing MSCs with CD34＋ cells iso- lated from cord blood. Our results showed that MSCs separated from these four different sections including UC, W J, UV, and AM had similar biological characteristics. All of the MSCs had multi-lineage differentiation ability and were able to differentiate into osteoblasts, adipocytes, and chondrocytes. The MSCs also inhibited the proliferation of allogeneic T cells in a dose-dependent manner. The relative mRNA expression of cytokines was examined, and the results showed that UCMSCs had higher interleukin-6 （IL6）, ILll, stem cell factor, and FLT3 expression than MSCs derived from specific sections of UCs. CD34＋ cells had high propagation efficiencies when co-cultured with MSCs derived from different sections of UCs, among which UCMSCs are the most efficient feeding layer. Our study demonstrated that MSCs could be isolated from whole UC or specific sections of UC with similar immuno- modulation and hematopoiesis supporting characteristics.