Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

Introduction

Human fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.

Methods

Human fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.

Results

The addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.

Conclusion

The inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Use of human albumin as an additional cryoprotective agent in freeze preservation of hematopoietic stem cells

14 bone-marrow samples of healthy donors were cryopreserved with 5% of DMSO in combination with different volume percentages of 20% human albumin, with the protective impact of the respective freezing mixture on the proliferating capacity of early hemotopoietic precursor cells being determined by means of CFU-c-technique. A combination of 5% of DMSO with 20% of human albumin with an addition of 20% of autologous serum proved to be the most favourable freezing protection. CFU-c-recovery which was achieved in this way amounted to 78 +/- 7%. A protective impact of human albumin for CFU-c during incubation with the freezing medium at room temperature was statistically not significant. Thus, the method presented is suitable for cryopreservation of bone marrow for autologous transplantation. The advantages over the traditional procedure results from the slight degree of toxicity by reducing the DMSO percentage from the common 10% to 5% as well as from the fact that the thawed cell suspension may be directly infused with the substance for freezing protection, thus avoiding an additional loss of proliferative material. The procedure may be used especially for autologous transplantation in childhood.     

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Adipose tissue as a potential source of hematopoietic stem/progenitor cells

It has been more than 30 years since adipose tissue (AT) has been recognized as a central modulator orchestrating sophisticated process termed "immunometabolism". Nonetheless, despite its unique involvement in the regulation of immune and endocrine homeostasis, recent studies demonstrated that AT also contains significant number of hematopoietic stem/progenitor cells (HSPCs) that may be there "settling down" throughout life. In this article we will focus on presenting the current concepts regarding endocrine, immunological, and molecular mechanisms that may contribute to and regulate bone marrow (BM)-derived HSPCs homing into AT environment, as well as, highlight various structural and morphological similarities between BM and AT that might be involved in creating appropriate tissue niches for BM-derived HSPCs in AT. Finally, we will discuss how development of obesity or type 2 diabetes may influence balance of homing signals for HSPCs in AT environment.     

Immature hematopoietic stem cells undergo maturation in the fetal liver

Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Active RHOA favors retention of human hematopoietic stem/progenitor cells in their niche

Background

Hematopoietic stem/progenitor cells (HSPCs) maintain the hematopoietic system by balancing their self-renewal and differentiation events. Hematopoietic stem cells also migrate to various sites and interact with their specific microenvironment to maintain the integrity of the system. Rho GTPases have been found to control the migration of hematopoietic cells and other cell types. Although the role of RAC1, RAC2 and CDC42 has been studied, the role of RHOA in human hematopoietic stem cells is unclear.

Results

By utilizing constitutively active and dominant negative RHOA, we show that RHOA negatively regulates both in vitro and in vivo migration and dominant negative RHOA significantly increased the migration potential of human HSC/HPCs. Active RHOA expression favors the retention of hematopoietic stem/progenitor cells in the niche rather than migration and was found to lock the cells in the G0 cell cycle phase thereby affecting their long-term self-renewal potential.

Conclusion

The current study demonstrates that down-regulation of RHOA might be used to facilitate the migration and homing of hematopoietic stem cells without affecting their long-term repopulating ability. This might be of interest especially for increasing the homing of ex vivo expanded HSPC.     

Expression of polypeptide GalNAc-transferases in hematopoietic stem/progenitor cells

The members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family, which transfer GalNAc to polypeptide serine and threonine residues, initiate mucin-type O-linked glycosylation. There are at least 13 functionally characterized members of this family in humans, but no studies have been reported of pp-GalNAc-T isoforms in hematopoietic cells. We isolated and purified CD34+ hematopoietic cells from adult bone marrow by magnetic cell sorting and induced them to differentiate into megakaryocytic lineage cells using an optimal combination of hematopoietic growth factors in serum-free liquid medium. RT-PCR revealed that CD34+ cells expressed pp-GalNAc-T1, T2, T3, T4, T6, T7, T10, T11 and T14, but not pp-GalNAc-T8, T9, T12 and T13. The megakaryocytic lineage cells showed significant increases in the expression of pp-GalNAc-T3, T8, T9, T10 and T13, but pp-GalNAc-T11 and T14 became undetectable. In summary, many pp-GalNAc-T isoforms were expressed in CD34+ cells but the expression pattern changed during differentiation into megakaryocytes. The expression patterns of pp-GalNAc-Ts may be necessary to ensure proper O-glycosylation of mucin-type proteins expressed in CD34+ and megakaryocytic cells.     

Identification of differentially expressed genes in epithelial stem/progenitor cells of fetal rat liver

Differentially expressed cDNA clones from fetal rat liver were isolated using suppression subtractive hybridization, combined with an efficient screening strategy. Approximately 30,000 clones were screened, yielding 643 genes whose expression was induced, of which 201 clones were distinct and 68 represented ESTs or newly discovered genes of unknown function. Based on their expression patterns in different organs, fetal liver, liver regeneration models, and gut epithelial progenitor cell lines, the subtracted clones presented in this work were placed into four categories: (1) hepatoblast-specific genes; (2) hematopoietic cell-specific genes; (3) genes expressed in hepatoblasts, in hematopoietic cells, and at varying levels in other tissues; and (4) genes overexpressed in fetal liver, in models of activation of liver progenitor cells, and in epithelial progenitor cell lines. Hepatoblast-specific clones and those representing genes induced during liver regeneration are under further study to define their specific function(s) in liver cell growth control and/or differentiation.     

Detection of cytokines in supernatant from hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and endothelial progenitor cells

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.     

Endogenous DNA damage clusters in human hematopoietic stem and progenitor cells

Clustered DNA damages-multiple oxidized bases, abasic sites, or strand breaks within a few helical turns-are potentially mutagenic and lethal alterations induced by ionizing radiation. Endogenous clusters are found at low frequencies in unirradiated normal human cells and tissues. Radiation-sensitive hematopoietic cells with low glycosylase levels (TK6 and WI-L2-NS) accumulate oxidized base clusters but not abasic clusters, indicating that cellular repair genotype affects endogenous cluster levels. We asked whether other factors, i.e., in the cellular microenvironment, affect endogenous cluster levels and composition in hematopoietic cells. TK6 and WI-L2-NS cells were grown in standard medium (RPMI 1640) alone or supplemented with folate and/or selenium; oxidized base cluster levels were highest in RPMI 1640 and reduced in selenium-supplemented medium. Abasic clusters were low under all conditions. In primary hematopoietic stem and progenitor cells from four non-tobacco-using donors, cluster levels were low. However, in cells from tobacco users, we observed high oxidized base clusters and also abasic clusters, previously observed only in irradiated cells. Protein levels and activity of the abasic endonuclease Ape1 were similar in the tobacco users and nonusers. These data suggest that in highly damaging environments, even normal DNA repair capacity can be overwhelmed, leaving highly repair-resistant clustered damages.     

New relationships of human hematopoietic lineages facilitate detection of multipotent hematopoietic stem and progenitor cells

Three important goals of hematopoietic stem cell research are to understand of how hematopoietic stem cells (HSCs) self-renew, how lineage commitment takes place, and how HSCs can be expanded ex vivo. Research in this area requires a reliable model of hematopoiesis. Performing detailed functional analyses of human hematopoietic progenitor subsets, we recently gained evidence for new hematopoietic lineage relationships.¹ According to our data, neutrophils belong to the same branch of the hematopoietic tree as lymphocytes. In contrast, eosinophils and basophils derive from another branch, the erythro-myeloid branch. Here, after introducing the newly proposed hematopoietic model, we discuss its consequences for the identification and expansion of human multipotent progenitors and suggest a fast and reliable method to screen for multipotent hematopoietic cells in vitro.     

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.     

Temporal molecular program of human hematopoietic stem and progenitor cells after birth

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Lineage development of hematopoietic stem and progenitor cells

Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.