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.     

Effective expansion of umbilical cord blood hematopoietic stem/progenitor cells by regulation of microencapsulated osteoblasts under hypoxic condition

The expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB) with the support of microencapsulated osteoblasts under hypoxia environment was investigated. The expansion of HSPCs was evaluated through the total number of UCB mononuclear cells (MNCs) produced, their repopulating potential with the colony-forming unit assay (CFU-Cs) and CD34⁺ phenotypic analysis with flow cytometry. At the end of 7 days of culture, the UCB-MNCs, CFU-Cs and CD34⁺ cells had achieved 18.7 ± 1.6, 11.6 ± 0.9 and 23.4 ± 2-fold expansions, respectively, in the test groups. These were significantly different from those in control groups. Microencapsulated osteoblasts under hypoxia conditions had therefore a significant effect on the expansion potential of HSPCs in vitro.     

Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self‐renewal. In fact, long‐term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O₂ concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O₂ solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O₂ concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34⁺ cells from umbilical cord blood. The stirring system was designed on top of a standard six‐well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O₂ for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c‐kit expression of CD34⁺ cells at 5% O₂, using 21%‐O₂ cultures as a control. In hypoxia we found that CD34⁺ cells maintained a higher expression of c‐kit. Further investigation is needed to explore the dynamics of interaction between oxygen‐ and c‐kit‐dependent pathways at the molecular level. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

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.     

Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34⁺ cells but also increases CD34⁻ cell numbers. These CD34⁻ cells influenced the ex vivo expansion of CD34⁺ cells. In this work, the effects of periodically removing CD34⁻ cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34⁻ cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34⁺ cells in the CD34⁻ cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34⁻ cells with SR1 addition improved the biological function of expanded CD34⁺ cells and significantly increased the percentage of self‐renewal symmetric division of CD34⁺ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34⁻ cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34⁻ cells plus cooperation with SR1 improved the expansion of CD34⁺ cells, maintained better biological function of expanded CD34⁺ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

SR1 increased self‐renewal symmetric division of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 increased ex vivo expansion of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 further downregulated AhR signalling     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a “biomimetic niche.” The CD34⁺ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture, the CD34⁺ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34⁺ cells. CD34⁺/CD38⁻ cells accounted for 82–90% of CD34⁺ cells. After 5 weeks, CD34⁺/CD38⁻ cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 10³ vs. 1.0 ± 0.5 × 10⁴, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 10³ vs. 2.5 ± 0.7 × 10², p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds) after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34⁺ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34⁺ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34⁺ HSPCs were quadratic inversely correlated with the Dcx index (r = ?0.5343 and r = ?0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34⁺ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.     

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal‐based serum‐free culture system to evaluate the effect of different initial CD34⁺ cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34⁺ and CD34⁺CD90⁺ expression, we have identified early activation of CD34 expression on CD34^? cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34⁺ cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34⁺/CD34⁺CD90⁺ cell yield (High: 14 ± 1.0/3.5 ± 1.4‐fold; Medium:22 ± 2.0/3.4 ± 1,0‐fold; Low:31 ± 3.0/4.4 ± 1.5‐fold) after a 7‐day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34⁺ cell recovery for each strategy, on overall CD34⁺ cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34⁺ cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10⁶ cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex‐vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost‐effective expansion of HSC for cellular therapy. J. Cell. Biochem. 112: 1822–1831, 2011. © 2011 Wiley‐Liss, Inc.     

The human placenta is a hematopoietic organ during the embryonic and fetal periods of development

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations—CD34⁺⁺CD45^low and CD34⁺CD45^low—that were found in chorionic villi and in the chorioamniotic membrane. CD34⁺⁺CD45^low cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34⁺⁺CD45^low cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56⁺ natural killer cells and CD19⁺CD20⁺sIgM⁺ B cells in polyclonal liquid cultures. CD34⁺CD45^low cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34⁻ cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34⁺⁺CD45^low cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.     

Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34⁺ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin^-CD34⁺CD38^lowCD45RA^-CD90⁺ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC.     

Higher efficacy of Etoposide + Cytarabine Plus Pegfilgrastim in poorly mobilizing Multiple Myeloma and lymphoma Patients

Background aimsAn optimal strategy for mobilizing hematopoietic stem cells in poorly mobilizing patients with multiple myeloma (MM) and lymphoma has not yet been determined.MethodsWe retrospectively analyzed the efficacy and safety of etoposide combined with cytarabine (etoposide 75 mg/m², daily d1∼2; Ara-C 300 mg/m², every 12 h d1∼2), plus pegfilgrastim (6 mg d6) in 32 patients with MM or lymphoma, among whom 53.1% were defined as “proven poor mobilizers.”ResultsThis approach resulted in adequate mobilization (≥2.0 × 10⁶ CD34⁺ cells/kg) in 93.8% of patients and optimal mobilization (≥5.0 × 10⁶ CD34⁺ cells/kg) in 71.9% of patients. A total of 100% of patients with MM reached at least 5 × 10⁶ CD34⁺ cells/kg collected, the amount required for double autologous stem cell transplant. In total, 88.2% of patients with lymphoma reached at least 2 × 10⁶ CD34⁺ cells/kg collected, the amount required for a single autologous stem cell transplant. This was achieved with a single leukapheresis in 78.1% of cases. A median peak number of 42.0/μL circulating CD34⁺ cells and a median number of blood CD34⁺ cells counts in 6.7 × 10⁶/L were collected among 30 successful mobilizers. Approximately 6.3% of patients required plerixafor rescue, which was successful. Nine (28.1%) of the 32 patients suffered grade 2∼3 infections, and 50% required platelet transfusions.ConclusionsWe conclude that chemo-mobilization with etoposide, Ara-C and pegfilgrastim in poorly mobilizing patients with MM or lymphoma is very effective and has acceptable toxicity.     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增。在该生物反应器内, 采用SCF+TPO+Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34+细胞的效果。培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34+细胞扩增倍数、培养物中CD34+细胞含量均相近, 无显著性差异; 而CD34+CD38-细胞扩增倍数以及培养物中CD34+CD38?细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养。可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增。     

一种新型生物反应器在造血干/祖细胞体外扩增中的应用

针对造血干/祖细胞体外扩增对培养环境的需求, 结合静/动态培养的特点, 开发了一种新型的生物反应器用于造血干/祖细胞的体外扩增.在该生物反应器内, 采用SCF TPO Flt-3细胞因子组合, 比较了静态和循环培养两种方式体外扩增脐血CD34 细胞的效果.培养7 d后, 总细胞分别扩增了(13.86 ± 4.26)和(7.23 ± 2.67)倍, 显示静态培养有利于总细胞的扩增; CD34 细胞扩增倍数、培养物中CD34 细胞含量均相近, 无显著性差异; 而CD34 CD38-细胞扩增倍数以及培养物中CD34 CD38-细胞的百分含量分别为(1.82 ± 0.58)和(3.90 ± 0.85)倍以及(9.45 ± 4.85)和(37.47 ± 14.06)%, 循环培养明显高于静态培养.可见, 在该生物反应器内, 采用静态和循环两种培养方式, 均能实现造血干/祖细胞的体外扩增, 但静态培养促使造血干细胞向定向祖细胞分化, 而循环培养则更有利于早期造血干细胞的扩增.     

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.     

Mobilization and engraftment of peripheral blood stem cells in healthy related donors >55 years old

Background aimsThe increasing scarcity of young related donors has led to the use of older donors for related allogeneic hematopoietic stem cell transplantation (HSCT). This study analyzed the influence of age on the results of mobilization of peripheral blood stem cells (PBSCs) in healthy donors as well as on the engraftment and outcome of HSCT.MethodsA retrospective analysis from a single center was performed comparing the results of PBSC mobilization from related healthy donors according to their age.ResultsThe study included 133 consecutive related donors. The median age was 50 years (range, 4–77 years); 70 (53%) donors were males, and 44 (33%) were >55 years old. All donors were mobilized with granulocyte colony-stimulating factor for 5 days. The peak CD34⁺ cell count in peripheral blood was higher in younger than in older donors (median, 90.5 CD34⁺ cells/μL [range, 18–240 CD34⁺ cells/μL] versus 72 CD34⁺ cells/μL [range, 20–172.5 CD34⁺ cells/μL], P = 0.008). The volume processed was lower in younger than in older donors (16,131 mL [range, 4424–36,906 mL] versus 18,653 mL [range, 10,003–26,261 mL], P = 0.002) with similar CD34⁺ cells collected (579.3 × 10⁶ cells [range, 135.14 × 10⁶–1557.24 × 10⁶ cells] versus 513.69 × 10⁶ cells [range, 149.81 × 10⁶–1290 × 10⁶ cells], P = 0.844). There were no differences in time to recovery of neutrophils and platelets or in the incidences of acute and chronic graft-versus-host disease, overall survival, non-relapse mortality and relapse incidence.ConclusionsDonors >55 years old mobilized fewer CD34⁺ cells and required a greater volume to collect a similar number of CD34⁺ cells. The outcome of HSCT was not influenced by donor age. Donor age should not be a limitation for related allogeneic HSCT.