Successful generation of donor specific hematopoietic stem cell lines from co-cultured bone marrow with human embryonic stem cell line: a new methodology

We report the generation of 30 healthy human embryonic stem cell (h-ESC) lines from 33 voluntary oocyte donors using a donor somatic cell nuclear transfer (SCNT) technique on 190 oocytes. Our aim was to coculture them with their own bone marrow (BM) to generate hematopoietic progenitor cells for therapeutic purposes. Pluripotency and undifferentiated stage were confirmed using molecular cell surface markers. Normal karyotype of these cell lines was confirmed. Here we demonstrate that SCNT-h-ESCs differentiate to hematopoietic precursors when cocultured with unmodified, nonirradiated donor BM. We did not use any xenogeneic material for this hematopoietic differentiation. Hematopoietic precursors derived from them expressed cell surface antigens CD45/34. When further cultured with hematopoietic growth factors these hematopoietic precursors formed characteristic myeloid, erythroid, and megakaryocyte lineages. Phenotypic CD34+ cells derived from NT-h-ESCs were functionally similar to their counterparts in primary hematopoietic tissues like BM, umbilical cord, and blood. More terminally differentiated hematopoietic cells derived from h-ESCs under these culture conditions also expressed normal surface antigens like glycophorin A on erythroid cells, CD15 on myeloid cells, and CD41 on megakaryocytes. We report generation of hematopoietic progenitor cells from h-ESC lines by a SCNT technique, with differentiation into further lineages with structural and functional similarities to their adult counterparts in vivo. This novel alternative source of CD34+ stem cells from h-ESC lines generated without any xenogeneic material might be used to create transplantation tolerance, to implement regenerative medicine, and to treat autoimmune disorders.     

Effects of flt-3 ligand in combination with TPO on the expansion of megakaryocytic progenitors

As an early acting growth factor, flt-3 ligand (FL) promotes the ex vivo expansion of hematopoietic stem and progenitor cells. The effect and mechanism of FL on the development of the megakaryocytic lineage remain unclear. In this study, we compared the effects of FL and stem cell factor (SCF) in combination with other megakaryocyte-promoting cytokines on the differentiation and proliferation of megakaryocytic progenitors and investigated the expression of flt-3 receptors on megakaryocytic cell lines. In liquid cultures of enriched CD34+ cells from human umbilical cord blood for 14 days, FL plus thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6 promoted the expansion of nucleated cells, CD34+ cells, CD34+ CD38- cells, and megakaryocyte colony-forming units (CFU-MK) by 300 +/- 115-, 23.8 +/- 11.3-, 33.9 +/- 28.6-, and 584 +/- 220-fold, respectively. Replacing FL with SCF significantly decreased the yield of all cell types. Using murine bone marrow (BM) cells, we demonstrated that FL at a range of 0-100 ng/ml had no significant mitogenic effect on CFU-MK formation. TPO increased CFU-MK (p < 0.001) but the effect was not significantly modified by FL. While one human acute lymphoblastic leukemia sample expressed high levels of flt-3 receptor, the four megakaryocytic cell lines (Meg-01, CHRF-288-11, M-07e, and Dami) did not show any positive expression. Our data suggest that the present cytokine combination and expansion conditions provide an effective and potentially useful system for the clinical expansion of cord blood for bone marrow transplantation (BMT). FL alone did not stimulate megakaryocytopoiesis, possibly due to the lack of receptor expression on megakaryocytes. The effect of FL in augmenting the expansion of CFU-MK in liquid culture might be due to the early action of FL at the pluripotent stem cell stage.     

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

RANK expression in vivo on hematopoietic subsets including pre-osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14(+)RANK(high) cells constitute a circulating pre-osteoclast pool. INTRODUCTION: The expression of RANK by subsets of hematopoietic cells has not been adequately studied in humans. While attributed to the monocytoid lineage, the phenotype of the pre-osteoclast (pre-OC) with respect to RANK expression in vivo remains unclear. We tested monoclonal antibodies (MAbs) raised against the extracellular domain of recombinant human RANK for reactivity with normal peripheral blood (PB) and bone marrow (BM) mononuclear cells (PBMNCs and BMMNCs, respectively). We also tested reactivity with giant cell tumor cells (GCT), a confirmed source of pre-OC and mature OCs. MATERIALS AND METHODS: Human PBMNCs, BMMNCs, and GCT cells were analyzed for reactivity with anti-RANK MAbs by flow cytometry in combination with hematopoietic lineage restricted markers. GCTs were also analyzed by immunofluorescence. CD14+ monocytoid cells were sorted by fluorescence-activated cell sorting (FACS) based on their relative RANK expression and cultured under OC-forming conditions. RESULTS: RANK+ cells were detected similarly by three independent anti-RANK MAbs. One MAb (80736) immunoprecipitated RANK-RANKL complexes from surface-biotinylated GCT lysates. Using dual-color flow cytometry, RANK was detected on CD14+ (monocytoid), CD19+ (B-lymphoid), CD56+ (NK cell), and glycophorin A+ erythroid progenitors. Minor populations of both CD3+ T lymphocytes and BM CD34+ hematopoietic progenitors also expressed cell surface RANK. In GCTs, RANK expression was identified on mononuclear CD45(+)CD14(+)alphaVbeta3(+)c-Fms+ cells, likely to be committed pre-OC, and on multinucleated CD45(+)alphaVbeta3(+)TRACP(+) OCs. Importantly, sorted CD14(+)RANK(high) PBMNCs treated with recombinant RANKL and macrophage-colony stimulating factor (M-CSF) gave rise to approximately twice the number of osteoclasts than RANK(mid) or RANK(low) cells. CONCLUSIONS: These results suggest that committed monocytoid RANK+ pre-OCs are represented in the marrow and circulate in the periphery, forming a pool of cells capable of responding rapidly to RANKL. The ability to reliably detect committed pre-OC in peripheral blood could have important clinical applications in the management of diseases characterized by abnormal osteoclastic activity.     

Identification of multiple osteoclast precursor populations in murine bone marrow.

Murine BM was fractionated using a series of hematopoietic markers to characterize its osteoclast progenitor populations. We found that the early osteoclastogenic activity in total BM was recapitulated by a population of cells contained within the CD11b(-/low) CD45R- CD3- CD115high fraction. INTRODUCTION: Osteoclasts are of hematopoietic origin and they have been shown to share the same lineage as macrophages. We further characterized the phenotype of osteoclast progenitor populations in murine bone marrow (BM) by analyzing their cell surface markers. MATERIALS AND METHODS: We used fluorescence-activated cell sorting (FACS) to identify the subsets of BM cells that contained osteoclast progenitors. We fractionated BM according to several markers and cultured the sorted populations for a period of 2-6 days with macrophage-colony stimulating factor (M-CSF) and RANKL. The numbers of multinucleated osteoclast-like cells (OCLs) that formed in the cultures were counted. RESULTS: We found that the CD45R- CD11b(-/low) population recapitulated the early osteoclastogenic activity of total BM. In addition, although previous experiments indicated that osteoclastogenic activity was enriched within the CD45R+ population, we found that highly purified CD45R+ BM was incapable of differentiating into osteoclasts in vitro. We also found that CD45R- CD11b(high) BM cells were an inefficient source of osteoclast progenitors. However, CD11b was transiently upregulated by cells of the CD45R- CD11b(-/low) fraction early (within 24 h) during culture with M-CSF. Finally, further fractionation of BM using CD115 and CD117 showed that, as osteoclast precursor cells matured, they downregulate CD117 but remain CD115+. Curiously, pure populations of CD117- (CD115high) cells isolated fresh from BM have low osteoclastogenic activity in vitro. CONCLUSIONS: We provided a refined analysis of the precise subpopulations of murine BM that are capable of differentiating into OCLs in vitro when treated with M-CSF and RANKL.     

人脐血造血干/祖细胞的磁力搅拌悬浮培养及移植实验

目的 探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法 从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论 磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.     

Characterization of thrombopoietin kinetics within 60 days after allogeneic hematopoietic stem cell transplantation and its correlation with megakaryocyte ploidy distribution

Thrombopoietin (TPO) has been identified as a key cytokine for both megakaryogenesis and thrombopoiesis. We attempt to characterize the kinetics of TPO and its correlation with megakaryocytes (MKs) ploidy distribution pattern within 60 d after allogeneic hematopoietic stem cell transplantation (allo‐HSCT). Forty‐six consecutive patients undergoing allo‐HSCT from October 2008 to December 2008 were included. TPO levels and ploidy distribution patterns of MKs were measured using ELISA and flow cytometric analysis, respectively. The results indicated that TPO levels and the platelet count followed opposite trends after allo‐HSCT. The preconditioning TPO levels and the number of transplanted CD34⁺ cells were significant predisposing factors for rapid platelet engraftment (p = 0.010 and 0.007, respectively) by multivariate analysis. There was a reduction of ploidy and an increase in immature MKs in patients with higher endogenous TPO levels (>250 pg/mL) on day 60 after allo‐HSCT. Moreover, lower TPO levels (≤250 pg/mL) on day 60 after allo‐HSCT were associated with significantly improved five‐yr overall survival (p = 0.021) and reduced transplant‐related mortality (p = 0.033). In conclusion, endogenous TPO levels may be associated with platelet recovery and have prognostic significance during allo‐HSCT.     

Impaired erythropoiesis after haemorrhagic shock in mice is associated with erythroid progenitor apoptosis in vivo

Objective: Multiply traumatised patients often suffer from blood loss and from subsequent therapy-resistant anaemia, possibly mediated by apoptosis, necrosis, or humoral factors. Therefore, the underlying mechanisms were investigated in bone marrow (BM) and peripheral blood in a murine resuscitated haemorrhagic shock (HS) model.
Methods: In healthy male mice, pressure-controlled HS was induced for 60 min. The BM was analysed for Annexin-V, 7-amino-actinomycin D, apoptotic enzymes (caspases-3/7, -8, and -9), expression of death receptors (CD120a, CD95), mitochondrial proteins (Bax, Bcl-2, Bcl-x), as well as erythropoietin (EPO) receptor (EPO-R). Blood cell count, peripheral EPO, and tumour necrosis factor-α response were additionally monitored.
Results: Twenty-four and 72 h after HS, EPO and EPO-R were strongly up-regulated in peripheral blood and BM, respectively. Decreasing numbers of erythroid progenitors in BM after HS correlated with significant apoptotic changes confirmed by increased caspases-3/7, -8, -9 activity in total BM, death receptor CD95 and CD120a expression on erythroid progenitors, and down-regulated mitochondrial Bcl-2 expression in total BM. Erythroid progenitors in peripheral blood were found to be increased after 72 h.
Conclusion: Despite the massive EPO response and up-regulation of EPO-R, BM erythroblasts (EBs) decreased. This could be due to deficient maturation of erythroid progenitors. Furthermore, the increased intrinsic and extrinsic apoptosis activation suggests programmed death of erythroid progenitors. We propose that both apoptosis and negatively regulated erythropoiesis contribute to BM dysfunction, while erythroid progenitor egress plays an additional role.     

人脐血造血干/祖细胞的生物反应器大规模扩增及移植实验

目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.     

Conditions that enable human hematopoietic stem cell engraftment in all NOD-SCID mice

BACKGROUND: Transplantation of human hematopoietic stem cells is the only true test of their long-term repopulation potential. Models are readily available to evaluate murine hematopoietic stem cells, but few exist that allow reliable quantification of human stem cells. The non-obese diabetic-severe combined immunodeficient (NOD-SCID) mouse model enables quantification of human hematopoietic stem cells, but the conditions that permit human engraftment in all animals have yet to be defined. The aims of the project were, therefore, to describe the variables that allow human engraftment in the NOD-SCID mouse model and the techniques that accurately quantify this engraftment. METHODS: NOD-SCID mice that had or had not received 250, 325, or 400 cGy irradiation received cord blood (CB) mononuclear or CD34+ cells i.v. or i.p. Mice were killed 6 weeks after transplantation, and the bone marrow, spleen, and thymus were harvested. Four-color flow cytometric analysis, semi-quantitative PCR, myeloid and erythroid progenitor, and stem cell assays were used to monitor human engraftment. RESULTS: A 250 or 325 cGy and i.v. injection of CB mononuclear or CD34+ cells is required to detect multilineage human engraftment in the bone marrow, spleen, or thymus of NOD-SCID mice. Four-color flow cytometric analysis and semi-quantitative PCR enable accurate detection of 0.1% human cells. Progenitor and stem cell assays provide functional information about the engrafted cells. CONCLUSIONS: Successful development of the NOD-SCID mouse model and techniques to assess human engraftment now allow it to be used reliably to analyze the effects of short-term cytokine exposure on the long-term repopulating capacity of CB stem cells.     

Preserved long-term repopulation and differentiation properties of hematopoietic grafts subjected to ex vivo expansion with stem cell factor and interleukin 11.

BACKGROUND: The ex vivo expansion of hematopoietic grafts has been proposed as an efficient procedure for improving the hematological recovery of recipients. The fate of the long-term repopulating cells during the ex vivo manipulation of the graft is, however, a critical issue in ex vivo expansion protocols and ultimately will define the applicability of this new technology in hematopoietic transplants. METHODS: The repopulating ability of mouse hematopoietic samples was determined by means of bone marrow (BM*) competition assays, using congenic strains that express the pan-leukocyte Ly-5.1 and Ly-5.2 antigens. The generation of potential changes in the repopulating properties of human hematopoietic samples subjected to ex vivo expansion was determined by comparing the engraftment of fresh and ex vivo-manipulated CD34+ cord blood cells in irradiated nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. RESULTS: Under our optimized conditions of mouse BM incubation (stem cell factor plus interleukin-11, either with or without macrophage inflammatory protein-1alpha or Flt3 ligand), both the short-term and the mid-term repopulating ability of the ex vivo-expanded samples were significantly improved when compared with fresh samples. In the long-term, no changes in the repopulation and differentiation properties of the graft were observed as a result of the ex vivo expansion process. As deduced from the analysis of NOD/SCID mice transplanted with fresh and ex vivo expanded human CD34+ cord blood cells, the in vitro stimulation mediated by SCF/IL-11/FLT3L was capable of preserving the ability of the grafts to repopulate the lympho-hematopoiesis of recipients for at least 3 months. CONCLUSION: These results indicate that under our optimized conditions of ex vivo expansion, the amplification of the hematopoietic progenitors responsible for the short- and mid-term repopulating properties of the graft can take place without compromising the long-term lympho-hematopoietic repopulating properties.     

Inhibitory effects of megakaryocytic cells in prostate cancer skeletal metastasis

Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC‐3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell‐cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC‐3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis‐associated specklike protein containing a CARD (ASC) and death‐associated protein kinase 1 (DAPK1) were increased in PC‐3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients. © 2011 American Society for Bone and Mineral Research.     

Beta-galactosidase of ROSA26 mice is a useful marker for detecting the definitive erythropoiesis after stem cell transplantation

BACKGROUND: Hematopoietic reconstitution after stem cell transplantation has been analyzed by using stem cells of Ly5 congenic mice. However, the early erythropoiesis has never been analyzed because this marker is not expressed on all of the erythroid lineage cells. The transgenic mouse expressing beta-galactosidase (beta-gal) or green fluorescent protein (GFP) has been reported. Using these markers, we analyzed the early erythropoiesis after stem cell transplantation. METHODS: The beta-gal activity and GFP were examined in the hematopoietic cells of ROSA26 and GFP transgenic mice, respectively, by flow cytometry. The primitive hematopoietic stem cell fraction (Lin(-)c-kit(+)Sca-1(+)) in bone marrow (BM) cells of ROSA26 mice was transferred into lethally irradiated mice. The kinetics of hematopoietic reconstitution was analyzed in the BM and spleen after transplantation. RESULTS: The beta-gal activity, but not the GFP and Ly5, was detected in all of the erythroid (TER119+) cells. The beta-gal activity was also detected in the donor-derived myeloid (Mac-1+), B lymphoid (B220+), and T lymphoid (Thy-1+) cells in the BM and spleen after stem cell transplantation. The kinetics of the hematopoietic reconstitution demonstrated that early erythroid (TER119(low)CD71(med)) cells were developed in the BM and spleen within 2 days after transplantation before development of proerythroblasts (TER119(+)CD71(high)), and that massive erythropoiesis and myelopoiesis were observed in the spleen until 2 and 4 weeks after transplantation, respectively. Conclusions. The beta-gal of ROSA26 mice can be a useful marker to identify the donor-derived hematopoietic cells, including early erythroid cells, and the first major wave of erythropoiesis occurring in the spleen after stem cell transplantation.     

Successful multilineage engraftment of human cord blood cells in pigs after in utero transplantation

BACKGROUND: Successful engraftment of human hematopoietic stem and progenitor cells (HSPCs) in a large animal may serve not only as a model to study human hematopoiesis but also as a bioreactor to expand human HSPCs in vivo. The aim of this study was to accomplish xenotransplantation of human HSPCs into pig. METHODS: Total mononuclear or CD34-positive HSPCs obtained from human cord blood were xenotransplanted percutaneously under an ultrasonographic guidance into preimmune pig fetuses. Peripheral blood and bone marrow (BM) cells of recipient pigs were collected and analyzed for the presence of human cells by a polymerase chain reaction to detect human specific Alu sequence on DNA extracted from those cells. Fluorescence-activated cell sorting (FACS) analysis was also performed to detect human hematopoietic cells. RESULTS: Transplantation of human cord blood cells into pig fetuses aged less than 52 days postcoitus resulted in a good engraftment rate. In one case, engraftment was detected up to 315 days posttransplantation by polymerase chain reaction. Human hematopoietic cells were detectable also by FACS in peripheral blood and BM. Furthermore, human CD34+ HSPCs were also observed in the BM of recipients. Those CD34+ cells in BM were sorted by FACS and subjected to further analyses. First, in vitro colony formation assay resulted in formations of multilineage colonies. Second, when they were transplanted into an immunodeficient mouse they were engrafted in the mouse. CONCLUSIONS: These data indicate an engraftment of human HSPCs in pig BM. In utero transplantation of human HSPCs into a preimmune pig fetus is useful to establish a pig reproducing human hematopoiesis.     

Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.     

Loss of Parathyroid Hormone Receptor Signaling in Osteoprogenitors Is Associated With Accumulation of Multiple Hematopoietic Lineages in the Bone Marrow

Osteoblasts and their progenitors play an important role in the support of hematopoiesis within the bone marrow (BM) microenvironment. We have previously reported that parathyroid hormone receptor (PTH1R) signaling in osteoprogenitors is required for normal B cell precursor differentiation, and for trafficking of maturing B cells out of the BM. Cells of the osteoblast lineage have been implicated in the regulation of several other hematopoietic cell populations, but the effects of PTH1R signaling in osteoprogenitors on other maturing hematopoietic populations have not been investigated. Here we report that numbers of maturing myeloid, T cell, and erythroid populations were increased in the BM of mice lacking PTH1R in Osx-expressing osteoprogenitors (PTH1R-OsxKO mice; knockout [KO]). This increase in maturing hematopoietic populations was not associated with an increase in progenitor populations or proliferation. The spleens of PTH1R-OsxKO mice were small with decreased numbers of all hematopoietic populations, suggesting that trafficking of mature hematopoietic populations between BM and spleen is impaired in the absence of PTH1R in osteoprogenitors. RNA sequencing (RNAseq) of osteoprogenitors and their descendants in bone and BM revealed increased expression of vascular cell adhesion protein 1 (VCAM-1) and C-X-C motif chemokine ligand 12 (CXCL12), factors that are involved in trafficking of several hematopoietic populations. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Differential hematopoietic supportive potential and gene expression of stroma cell lines from midgestation mouse placenta and adult bone marrow

During mouse embryogenesis, hematopoietic development takes place in several distinct anatomic locations. The microenvironment of different hematopoietic organs plays an important role in the proliferation and maturation of the hematopoietic cells. We hypothesized that fetal stromal cells would be distinct to adult bone marrow (BM)-derived stromal cells because the BM contributes mainly to the homeostasis of hematopoietic stem cells (HSCs), while extensive expansion of HSCs occurs during fetal development. Here we report the establishment of stromal cell lines from fetal hematopoietic organs, namely aorta-gonad-mesonephros (AGM), midgestation placenta (PL), and fetal liver (FL) together with adult bone marrow (BM). The growth patterns and hematopoietic supportive potential were studied. Their phenotypic and molecular gene expression profiles were also determined. Stromal cell lines from each tissue were able to support cobblestone area formation of BM c-Kit(+)Sca-1(+) hematopoietic cells: 22 (22/47) from AGM, three (3/4) from PL, three (3/4) from FL, and three (3/3) from BM. There were similar levels of expansion of total mononuclear cells (TMNs) when HSCs were cocultured with fetal stroma and adult BM stroma. However, PL-derived stromal cells supported higher levels of generation of colony-forming progenitor cell (CFU-C), indicated by more colonies and colonies with significantly larger size. Flow cytometric analysis of the PL1 cells demonstrated a phenotype of CD45(-), CD105(+), Sca-1(+), CD34(+), and CD49d(+), compared to adult BM1 cells, which were CD45(-), CD105(+), Sca-1(+), CD34(-), and CD49d(-). Using Affymetrix microarray analysis, we identified that genes specifically express in endothelial cells, such as Tie1, Tek, Kdr, Flt4, Emcn, Pecam1, Icam2, Cdh5, Esam1, Prom1, Cd34, and Sele were highly expressed in stroma PL1, consistent with an endothelial phenotype, while BM1 expressed a mesenchymal stromal phenotype. In summary, these data demonstrate distinct characteristics of stromal cells that provide insights into the microenvironmental control of HSCs.     

Clinical-scale cultures of cord blood CD34(+) cells to amplify committed progenitors and maintain stem cell activity

We developed a clinical-scale cord blood (CB) cell ex vivo procedure to enable an extensive expansion of committed progenitors--colony-forming cells (CFCs) without impairing very primitive hematopoietic stem cells (HSCs). CD34(++) cells, selected from previously cryopreserved and thawed CB units, were cultured in two steps (diluted 1:4 after 6 days) in the presence of stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt-3L), megakaryocyte growth and development factor (MGDF) (100 ng/ml each), granulocyte-colony stimulating factor (G-CSF) (10 ng/ml) in HP01 serum-free medium. HSC activity was evaluated in a serial transplantation assay, by detection of human cells (CD45, CD33, CD19 and CFC of human origin) in bone marrow (BM) of primary and secondary recipient NOD/SCID mice 6-8 weeks after transplantation. A wide amplification of total cells (～350-fold), CD34(+) cells (～100-fold), and CFC (～130-fold) without impairing the HSC activity was obtained. The activity of a particular HSC subpopulation (SRC(CFC)) was even enhanced.Thus, an extensive ex vivo expansion of CFCs is feasible without impairing the activity of HSCs. This result was enabled by associating antioxidant power of medium with an appropriate cytokine cocktail (i.e., mimicking physiologic effects of a weak oxygenation in hematopoietic environment).     

The effect of G-CSF-stimulated donor marrow on engraftment and incidence of graft-versus-host disease in allogeneic bone marrow transplantation

Graft-versus-host disease (GVHD) and infection are major obstacles to successful allogeneic bone marrow transplantation (allo-BMT). In an attempt to improve the results of HLA-identical sibling BMT, we investigated the effect of accelerating hemopoietic reconstitution and reducing acute GVHD (aGVHD) in allo-BMT receiving G-CSF-stimulated donor marrow and the preliminary biological mechanism. The donors of 30 patients (study group) with leukemia were given G-CSF 3-4 microg/kg/d for 7 doses prior to marrow harvest. The results of subsequent engraftment in the recipients were compared with those of 18 patients without G-CSF (control group). Five donors themselves were studied to assess the effects of G-CSF on the hematopoietic progenitor cells and lymphocyte subsets in the bone marrow (BM). We observed that the stimulated BM yielded higher numbers of nucleated cells as well as CFU-GM and CD34+ cells (p<0.01), and that hemopoietic reconstitution was accelerated. The median number of days of granulocyte count exceeding 0.5x10(9)/L and platelet count exceeding 20x10(9)/L was 16 (range 10-23 d) and 18.5 (range 13-31 d), respectively (control group: median 22 d, range 13-29 d and median 23 d, range 17-34 d; p=0.001). The incidence of grade II-IV severe aGVHD was very low, with only 1 case (3.3%) with acute grade II aGVHD limited to the skin in the study group. Five of 18 patients in the control group manifested grade II-IV severe aGVHD (27.8%, p=0.02). The number of T-lymphocyte subsets in the harvested BM using G-CSF stimulation was changed. In the G-CSF-stimulated marrow group, CD4+ decreased and CD8+ increased significantly (p=0.02). The changes of progenitor cells and T-lymphocyte subsets in donors' BM from pre- and post-G-CSF stimulation showed that the percentage of CD4+ reduced (p=0.04) and that of CD8+ increased (p=0.06), while that of CD34+ also increased (p=0.002). The incidence of chronic GVHD and relapse had no significant difference between both groups. These results indicate that allo-BMT in BM G-CSF priming can accelerate engraftment and minimize the incidence of severe aGVHD. There is a trend in favor of improved transplantation-related mortality.