Resveratrol improves ex vivo expansion of CB-CD34+ cells via downregulating intracellular reactive oxygen species level

Intracellular reactive oxygen species (ROS) play important roles in the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). In this study, the effects of resveratrol (RES), on the ex vivo expansion of HSPCs were investigated by analyzing CD34⁺ cells expansion and biological functions, with the objective to optimize ex vivo culture conditions for CD34 ⁺ cells. Among the five tested doses (0, 0.1, 1, 10, 20, and 50 μM), 10 μM RES was demonstrated to be the most favorable for ex vivo CD34 ⁺ cells expansion. In the primary cultures, 10 μM RES favored higher expansion folds of CD34 ⁺ cells, CD34 ⁺CD38 ⁻ cells, and colony-forming units (CFUs) ( P < 0.05). It was found that the percentages of primitive HSPCs (CD34 ⁺CD38 ⁻CD45R ⁻CD49f ⁺CD90 ⁺ cells) in 10 μM RES cultures were higher than those without RES. Further, in the secondary cultures, expanded CD34 ⁺ cells derived from primary cultures with 10 μM RES exhibited significantly higher total cells and CD34 ⁺ cells expansion ( P < 0.05). In the semisolid cultures, the frequency of CFU-GM and total CFUs of 10 μM RES group were both higher than those of without RES group, demonstrating that CD34 ⁺ cells expanded with 10 μM RES possessed better biological function. Furthermore, the addition of 10 μM RES downregulated the intracellular ROS level via strengthening the scavenging capability of ROS, and meanwhile reducing the percentages of apoptotic cells in cultures. Collectively, RES could stimulate the ex vivo expansion of CD34 ⁺ cells, preserved more primitive HSPCs and maintain better biological function by alleviating intracellular ROS level and cell apoptosis in cultures.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

CAPE promotes the expansion of human umbilical cord blood-derived hematopoietic stem and progenitor cells in vitro

Due to the low number of collectable stem cells from single umbilical cord blood(UCB)unit,their initial uses were limited to pediatric therapies.Clinical applications of UCB hematopoietic stem and progenitor cells(HSPCs)would become feasible if there were a culture method that can effectively expand HSPCs while maintaining their self-renewal capacity.In recent years,numerous attempts have been made to expand human UCB HSPCs in vitro.In this study,we report that caffeic acid phenethyl ester(CAPE),a small molecule from honeybee extract,can promote in vitro expansion of HSPCs.Treatment with CAPE increased the percentage of HSPCs in cultured mononuclear cells.Importantly,culture of CD34+HSPCs with CAPE resulted in a significant increase in total colony-forming units and high proliferative potential colony-forming units.Burst-forming unit-erythroid was the mostly affected colony type,which increased more than 3.7-fold in 1μg mL 1CAPE treatment group when compared to the controls.CAPE appears to induce HSPC expansion by upregulating the expression of SCF and HIF1-α.Our data suggest that CAPE may become a potent medium supplement for in vitro HSPC expansion.     

Efficient expansion of rare human circulating hematopoietic stem/progenitor cells in steady-state blood using a polypeptide-forming 3D culture

Although widely applied in treating hematopoietic malignancies, transplantation of hematopoietic stem/progenitor cells (HSPCs) is impeded by HSPC shortage. Whether circulating HSPCs (cHSPCs) in steady-state blood could be used as an alternative source remains largely elusive. Here we develop a three-dimensional culture system (3DCS) including arginine, glycine, aspartate, and a series of factors. Fourteen-day culture of peripheral blood mononuclear cells (PBMNCs) in 3DCS led to 125- and 70-fold increase of the frequency and number of CD34⁺ cells. Further, 3DCS-expanded cHSPCs exhibited the similar reconstitution rate compared to CD34⁺ HSPCs in bone marrow. Mechanistically, 3DCS fabricated an immunomodulatory niche, secreting cytokines as TNF to support cHSPC survival and proliferation. Finally, 3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization. Our 3DCS successfully expands rare cHSPCs, providing an alternative source for the HSPC therapy, particularly for the patients/donors who have failed in HSPC mobilization.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13238-021-00900-4.     

Characterization of human embryonic stem cell-derived hematopoietic progenitor phenotype

Differentiation of human embryonic stem cells (hESCs) into hematopoietic lineages using various methods has been reported. However, the phenotype that precisely defines the hematopoietic progenitor compartment with clonogenic activities has yet to be determined. Here, we measured and characterized progenitor function of subfractions of cells prospectively isolated from human embryoid bodies (hEBs) during hematopoietic differentiation basing on surface markers CD45, CD34, CD43, and CD38. We report that hematopoietic progenitors predominantly resided in the CD45⁺ subset. CD43⁺ cells lacking CD45 expression were largely devoid of progenitor activity. However, progenitor activity and multipotentiality was more enriched in CD45⁺ cells co-expressing CD43. CD45⁺ subset co-expressing CD34 but lacking CD38 expression (CD45⁺CD34⁺CD38^-) were further enriched for CFU capacity compared to the CD45⁺CD34⁺CD38⁺ subset. Our study demonstrates a role of CD43 in enriching hematopoietic progenitors derived from hEBs and reveals a hierarchical organization of hESC-derived hematopoietic progenitor compartments defined by phenotypic markers.     

Protein complexes associated with β-catenin differentially influence the differentiation profile of neonatal and adult CD8+ T cells

The canonical Wnt signaling pathway is a master cell regulator involved in CD8⁺ T cell proliferation and differentiation. In human CD8⁺ T cells, this pathway induces differentiation into memory cells or a “stem cell memory like” population, which is preferentially present in cord blood. To better understand the role of canonical Wnt signals in neonatal or adult blood, we compared the proteins associated with β-catenin, in nonstimulated and Wnt3a-stimulated human neonatal and adult naive CD8⁺ T cells. Differentially recruited proteins established different complexes in adult and neonatal cells. In the former, β-catenin-associated proteins were linked to cell signaling and immunological functions, whereas those of neonates were linked to proliferation and metabolism. Wnt3a stimulation led to the recruitment and overexpression of Wnt11 in adult cells and Wnt5a in neonatal cells, suggesting a differential connexion with planar polarity and Wnt/Ca²⁺ noncanonical pathways, respectively. The chromatin immunoprecipitation polymerase chain reaction β-catenin was recruited to a higher level on the promoters of cell renewal genes in neonatal cells and of differentiation genes in those of adults. We found a preferential association of β-catenin with CBP in neonatal cells and with p300 in the adult samples, which could be involved in a higher self-renewal capacity of the neonatal cells and memory commitment in those of adults. Altogether, our results show that different proteins associated with β-catenin during Wnt3a activation mediate a differential response of neonatal and adult human CD8⁺ T cells.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

Divergent effects of Wnt5b on IL-3- and GM-CSF-induced myeloid differentiation

The multiple specialized cell types of the hematopoietic system originate from differentiation of hematopoietic stem cells and progenitors (HSPC), which can generate both lymphoid and myeloid lineages. The myeloid lineage is preferentially maintained during ageing, but the mechanisms that contribute to this process are incompletely understood. Here, we studied the roles of Wnt5a and Wnt5b, ligands that have previously been linked to hematopoietic stem cell ageing and that are abundantly expressed by both hematopoietic progenitors and bone-marrow derived niche cells. Whereas Wnt5a had no major effects on primitive cell differentiation, Wnt5b had profound and divergent effects on cytokine-induced myeloid differentiation. Remarkably, while IL-3-mediated myeloid differentiation was largely repressed by Wnt5b, GM-CSF-induced myeloid differentiation was augmented. Furthermore, in the presence of IL-3, Wnt5b enhanced HSPC self-renewal, whereas in the presence of GM-CSF, Wnt5b accelerated differentiation, leading to progenitor cell exhaustion. Our results highlight discrepancies between IL-3 and GM-CSF, and reveal novel effects of Wnt5b on the hematopoietic system.     

Hematopoietic reconstitution of CD34<Superscript>+</Superscript> cells grown in static and stirred culture systems in NOD/SCID mice

The hematopoietic reconstitution of cord blood (CB) CD34⁺ cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.     

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.     

Bone marrow niche-inspired,multiphase expansion of megakaryocytic progenitors with high polyploidization potential

Background aimsMegakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization and platelet release. pH and pO₂ increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.MethodsCD34⁺ HSPCs were cultured at pH 7.2 and 5% O₂ with stem cell factor (SCF), thrombopoietin (Tpo) and all combinations of Interleukin (IL)-3, IL-6, IL-11 and Flt-3 ligand to promote Mk progenitor expansion. Cells cultured with selected cytokines were shifted to pH 7.4 and 20% O₂ to generate mature Mks, and treated with nicotinamide (NIC) to enhance polyploidization.ResultsUsing Tpo + SCF + IL-3 + IL-11, we obtained 3.5 CD34⁺ CD41⁺ Mk progenitors per input HSPC, while increasing purity from 1% to 17%. Cytokine cocktails with IL-3 yielded more progenitors and mature Mks, although the purities were lower. Mk production was much greater at higher pH and pO₂. Although fewer progenitors were present, shifting to 20% O₂/pH 7.4 at day 5 (versus days 7 or 9) yielded the greatest mature Mk production, 14 per input HSPC. NIC more than doubled the percentage of high-ploidy Mks to 40%.ConclusionsWe obtained extensive Mk progenitor expansion, while ensuring that the progenitors could produce high-ploidy Mks. We anticipate that subsequent optimization of cytokines for mature Mk production and delayed NIC addition will greatly increase high-ploidy Mk production.     

In vitro T lymphopoiesis: A model system for stem cell gene therapy for AIDS

Abstract: Stable introduction of therapeutic genes into hematopoietic stem cells has the potential to reconstitute immunity in individuals with HIV infection. However, many important questions regarding the safety and efficacy of this approach remain unanswered and may be addressed in a non-human primate model. To facilitate evaluation of expression of foreign genes in T cells derived from transduced hematopoietic progenitor cells, we have established a culture system that supports the differentiation of rhesus macaque and human CD34⁺ bone marrow derived cells into mature T cells. Thymic stromal monolayers were prepared from the adherent cell fraction of collagenase digested fetal or neonatal thymus. After 10–14 days, purified rhesus CD34⁺ bone marrow-derived cells cultured on thymic stromal monolayers yielded CD3⁺CD4⁺CD8⁺, CD3⁺CD4⁺CD8^?, and CD3⁺CD4^?CD8⁺ cells. Following stimulation with mitogens, these T cells derived from CD34⁺ cells could be expanded over 1,000-fold and maintained in culture for up to 20 weeks. We next evaluated the ability of rhesus CD34⁺ cells transduced with a retroviral vector containing the marker gene neo to undergo in vitro T cell differentiation. CD34⁺ cells transduced in the presence of bone marrow stroma and then cultured on rhesus thymic stroma resulted in T cells containing the retroviral marker gene. These studies should facilitate both in vitro and in vivo studies of hematopoietic stem cell therapeutic strategies for AIDS.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle

Background

Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization.

Results

Bull-derived CD34⁺ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34⁺ cells was lower in the freemartin than in its male siblings. Bull (Y⁺) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y⁺CD34⁺ cells, Y⁺CD34^- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation.

Conclusion

The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.