ALBERT

Description: Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p

Description: Platelets are released from terminally differentiated megakaryocytes (MKs) through lineage commitment, although the underlying mechanisms of megakaryopoiesis and subsequent thrombopoiesis are incompletely understood. MKs were reportedly generated from not only hematopoietic stem cells (HSCs), but also pre-adipocytes, without any gene transfer in an in vitro culture system (Matsubara et al, 2012 Methods Mol Biol). Because of high efficiency of platelet production from pre-adipocytes, much interest has been attracted to clarify the mechanisms of megakaryopoiesis from pre-adipocytes. Here we demonstrate a novel mechanism that megakaryopoiesis from pre-adipocytes is regulated by the inducible production of endogenous TPO via transferrin receptor CD71. NF-kB pathway in pre-adipocytes might modulate the TPO production through the stimulation of transferrin. Previously, we reported that pre-adipocyte cell line OP9 has the gene expressions of thrombopoietin (TPO) and its receptor c-MPL. This study first examined the TPO protein levels in supernatant from human (Cell Applications) and mouse pre-adipocytes cultured with MK lineage induction (MKLI) media (Ono et al, 2012 Blood, Matsubara et al, 2012 Methods Mol Biol) in the absence of recombinant TPO (rTPO). The TPO levels were 36.6 ± 9.2 pg/mL in mouse pre-adipocytes on Day 8 and 36.1 ± 5.8 pg/mL in human pre-adipocytes on Day 8, and undetectable levels of TPO were observed in both cells before MK induction. Differentiation efficiency (approximately 20%) of MKs from pre-adipocytes cultured with rTPO was similar to that without rTPO. The function of pre-adipocyte-derived platelets cultured with rTPO was similar to that without rTPO when assessed by incorporation of platelets into thrombus formation on the collagen-coated surface under flow conditions. The number of MKs produced from pre-adipocytes was markedly decreased in the presence of anti-c-MPL blocking antibody, AMM2. Regarding the MK differentiation from HSCs, it is well known that HSCs do not differentiate into MKs in the absence of rTPO. We did not observe the TPO secretion from human bone marrow CD34(+) cells and mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells in a culture condition using MKLI media in the absence of rTPO. The observations suggest that the MK differentiation from pre-adipocytes has a distinct mechanism of megakaryopoiesis from HSCs. These findings indicate the unique mechanism of megakaryopoiesis from pre-adipocytes; the endogenous TPO stimulation via c-MPL in pre-adipocytes has activity for promoting MK differentiation and subsequent production of functional platelets. To elucidate the molecular mechanism of TPO production during MK differentiation from pre-adipocytes, we examined which components of MKLI media were responsible for TPO production. We found iron-saturated transferrin, but not apo-transferrin, included in MKLI media is the critical factor to induce TPO production and MK differentiation from pre-adipocytes. The effects of transferrin were not observed when pre-adipocytes were cultured in the presence of anti-CD71 blocking antibody, 8D3, or deferoxamine mesilate, an iron-chelating agent. The effects of transferrin on TPO production and MK differentiation were also abolished in pre-adipocytes transfected with siRNA-CD71, and the TPO levels were 44.8 ± 15.9 pg/mL for siRNA-control and undetectable levels for siRNA-CD71 on Day 6. We next investigated a mechanism of inducing TPO production via CD71 in pre-adipocytes. The 5’ region of human TPO gene contains transcription factor-binding sequences, such as GATA1, GATA2 and NF-kB. Since NF-kB is a downstream target of CD71, we then examined whether NF-kB involved in MK differentiation derived from pre-adipocytes. Nuclear translocation of NF-kB in pre-adipocytes was observed when cells were stimulated with transferrin. Both TPO secretion and MK production were decreased in the presence of a peptide to inhibit nuclear translocation of NF-kB. Taken together, it is concluded that NF-kB pathway in pre-adipocytes modulates megakaryopoiesis through endogenous TPO production via CD71. The present findings suggest that pre-adipocytes have a unique mechanism of megakaryopoiesis through lineage commitment. Disclosures No relevant conflicts of interest to declare.

Description: Key Points GATA1 is downregulated in RPS19-deficient cells and zebrafish through upregulation of p53, TNF-α, and p38 MAPK. Treatment of rps19-deficient zebrafish with the TNF-α inhibitor etanercept rescues their erythroid and developmental defects.

Description: The clinical need for platelet transfusions is increasing; however, donor-dependent platelet transfusions are associated with practical problems, such as the limited supply and the risk of infection. Thus, we developed a manufacturing system for platelets from a donor-independent cell source: a human adipose-derived mesenchymal stromal/stem cell line (ASCL). The ASCL was obtained using an upside-down culture flask method and satisfied the minimal criteria for defining mesenchymal stem cells (MSCs) by The International Society for Cellular Therapy. The ASCL showed its proliferation capacity for ≥2 months without any abnormal karyotypes. The ASCL was cultured in megakaryocyte induction media. ASCL-derived megakaryocytes were obtained, with a peak at day 8 of culture, and ASCL-derived platelets (ASCL-PLTs) were obtained, with a peak at day 12 of culture. We observed that CD42b+ cells expressed an MSC marker (CD90) which is related to cell adhesion. Compared with peripheral platelets, ASCL-PLTs exhibit higher levels of PAC1 binding, P-selectin surface exposure, ristocetin-induced platelet aggregation, and ADP-induced platelet aggregation, as well as similar levels of fibrinogen binding and collagen-induced platelet aggregation. ASCL-PLTs have lower epinephrine-induced platelet aggregation. The pattern of in vivo kinetics after infusion into irradiated immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice was similar to that of platelet concentrates. ASCL-PLTs have similar characteristics to those of peripheral platelets and might have an additional function as MSCs. The establishment of the ASCL and its differentiation into ASCL-PLTs do not require gene transfer, and endogenous thrombopoietin is used for differentiation. The present protocol is a simple method that does not require feeder cells, further enhancing the clinical application of our approach.

Description: We have described the use of adipose tissue-derived stromal (ADS) cells (also known as pre-adipocytes) to generate megakaryocytes (MKs) and platelets (Plts) for potential application as non-donor-derived Plts in transfusion medicine. ADS cells are an attractive candidate cell source, because (1) their differentiation does not require gene transfer as they utilize endogenous genes in differentiation into functional MKs, (2) megakaryopoiesis and thrombopoiesis are dependent on endogenous thrombopoietin, and (3) ADS cells are genetically stable in long-term culture. However, Plt yield remains insufficient for clinical application. In the present study, we focused on the establishment and characterization of cell lines from small amount of human ADS cells (5 x 105 cells isolated from ~0.5 g fat). To clarify the usefulness of ADS cell line (ASCL) as a source for clinical application, characterization of ASCL and platelet function tests were performed on ASCL-derived MKs and Plts. We modified the established method, utilizing an upside-down culture flask method (Yagi et al, BBRC 2004), to obtain fat cell line with multipotent capacity. Instead of using large volume of fat as described in the original method, we developed ASCL from a small number of ADS cells, because ADS cells possess the capacity of expansion and differentiation into mature adipocytes rapidly. These ASCL have now been expanded in culture at least 6 months. Gene expression analysis by quantitative real-time PCR indicated that ASCL expressed the genes for several pluripotent cell-markers, such as, OCT3/4, KLF4, Myc, Nanog, Gal, and GABRB3. Also, ASCL expressed genes related to MK lineages, such as p45NF-E2, RUNX1, GATA2, Fli1, FOG1, TPO, and c-MPL. These phenotypes did not differ from ADS cells. These cells were then cultured in MK induction media consisting of IMDM containing transferrin. The frequency of CD41+/CD42b+ MK-sized cells on day 10 was 24 ± 2%. DNA polyploidy, a hallmark of MKs, in CD41+ cells ranged from 2N to 16N with an average of 4.3N. These results were similar to MKs derived directly from ADS cells. The frequency of CD41+/CD42b+ Plt-sized particles on day 18 was 34 ± 6%. For Plt yield, ~30 Plts were obtained from single ASCL. Analysis of granule contents showed that ASCL-derived Plts contain ATP and ADP, and von Willebrand factor. We next examined functionality of these ASCL-derived Plts. Donor Plts were used as a control. Upon stimulation with thrombin (0.5 U/mL), binding of labeled fibrinogen occurred to both ASCL-derived Plts and donor Plts. But binding to ASCL-derived Plts was higher (p

Description: We have established human adipose-derived mesenchymal stromal/stem cell line (ASCL) as an expandable cell source to generate megakaryocytes (MKs) releasing platelets for clinical transfusion. The use of ASCL has an advantage in manufacturing platelets, because both establishment of ASCL and its differentiation to MKs and platelets do not require gene transfer, and its endogenous thrombopoietin (TPO) is utilized for their differentiation. Here we report characterization of ASCL and ASCL-derived platelets for clinical application. ASCL retained their proliferation capacity for 6 months. There was no abnormality by karyotype analysis for ASCL, and no gene mutations registered in database for cancer and other diseases were noted by next-generation sequence analysis (n=3). ASCL satisfies the minimal criteria for defining mesenchymal stem cells by The International Society for Cellular Therapy: (1) adherence to plastic in vitro culture, (2) expression (〉95% of cells) of CD73, CD90, and CD105 and no expression (

Description: Diamond-Blackfan Anemia (DBA) is a rare inherited bone marrow failure disorder, characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. While several studies have found that the tumor suppressor protein p53 contributes to DBA pathogenesis, and that certain GATA1 mutations can give rise to DBA, the link between ribosomal protein mutations and erythroid defects is not well understood. To investigate the molecular pathways downstream of RPS19 deficiency, we infected human cord blood CD34+ cells with RPS19 shRNA lentivirus and observed that RPS19 knockdown resulted in decreased GATA1 mRNA and protein expression, resulting in defective erythropoiesis. We also detected increased TNF-α levels in RPS19 knockdown cells, and in zebrafish that have been treated with RPS19 morpholino. TNF-α was primarily upregulated in the non-erythroid population of RPS19 deficient cells, while its receptor, TNFR1, showed increased expression on the surface of erythroid cells. To understand the relationship between GATA1 and TNF-α, we treated primary hematopoietic CD34+ cells with TNF-α or its inhibitor etanercept. Our results demonstrated that TNF-α treatment reduced GATA1 expression, and that this effect could be rescued by the addition of etanercept. Treatment of RPS19 deficient cells with etanercept improved their erythroid differentiation, and increased GATA1 expression. Additionally, etanercept treatment successfully reversed the anemia phenotype observed in RPS19 deficient zebrafish. To study pathways downstream of TNF-α, we examined phosphorylation of signaling pathways such as p38 MAPK, NFκB, ERK, STAT1, and STAT5 in RPS19 deficient erythroid cells using phospho-flow cytometry. Among these pathways, we found a significant increase in phosphorylation of p38 MAPK, but not ERK, NFκB, STAT1, or STAT5, suggesting that p38 MAPK activation by TNF-α contributes to decreased GATA1 expression in RPS19 cells. We suggest a novel mechanism for the erythroid defects observed in DBA, in which RPS19 deficiency leads to increased TNF-α production in non-erythroid cells, and activation of p38 MAPK, followed by decreased GATA1 expression, in erythroid cells. Our data also suggest that TNF-α inhibitors, such as etanercept, may be beneficial in treating patients with DBA. Disclosures No relevant conflicts of interest to declare.