Hemopoietic Progenitor Cells Research Articles

Diagnosis and management of childhood aplastic anaemia

Aplastic anaemia (AA) is a rare and heterogeneous disorder. AA results in pancytopenia and a hypocellular bone marrow in the absence of an abnormal infiltrate, major dysplasia or marrow fibrosis. In children, most cases are idiopathic and caused by T lymphocyte-mediated destruction of haemopoietic stem and progenitor cells (HSPC's). Inherited bone marrow failure syndromes (IBMFS) account for around 20% of cases and have to be excluded. This can be challenging but has specific implications for management. Haemopoietic stem cell transplantation (HSCT) is the only definitive curative treatment for AA. For patients less than 35 years old with severe aplastic anaemia (SAA), a matched sibling donor (MSD) haematopoietic stem cell transplant is the treatment of choice. For those lacking such a donor, immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and ciclosporin has historically been initial treatment. Improved outcomes following matched unrelated donors (MUD) transplantation has led to UK guidelines recommending upfront MUD HSCT in children and young adults where a suitable donor can be quickly identified. Recent advances in the treatment of patients with AA have shown that horse ATG with cyclosporine remains the current standard IST. The thrombopoietin receptor agonist eltrombopag has significant activity as a single agent and in combination with IST as initial treatment and in refractory patients. For patients with IBMFS, transplantation remains the only curative procedure.

Targeting the niche: depleting haemopoietic stem cells with targeted therapy.

Haemopoietic stem cell transplantation is an expanding procedure worldwide but is associated with significant morbidity and mortality. Depletion of resident haemopoietic stem and progenitor cells (HSPC) is required for both autologous and allogeneic haemopoietic stem cell transplantation. Current conditioning protocols utilise chemotherapy or radiation to effectively reduce HSPC but are toxic in both the short and long term. The initial trials to use monoclonal antibodies to target HSPC were limited with marginal efficacy but platforms including antibody drug conjugates and chimeric antigen receptor T cells have made targeted conditioning strategies achievable. In this review we summarise the work developing targeted conditioning that may replace or reduce alkylating agents and total body irradiation. The prospect of conditioning with significantly reduced toxicity will improve outcomes and open transplantation to patients unable to tolerate current conditioning protocols.

Compatibility effects of ginseng and Ligustrum lucidum Ait herb pair on hematopoietic recovery in mice with cyclophosphamide-induced myelosuppression and its material basis

BackgroundGinseng (G) and Ligustrum lucidum Ait (LLA) are core traditional Chinese medicines in treating myelosuppression formula. The present study was designed to profile effect of G and LLA herb pair (G-LLA) on myelosuppressed mice. MethodsThe mice myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (Cy). Hematopoietic function of bone marrow was measured by hemopoietic progenitor cell culture and peripheral blood count, and serum hemopoietic factors were tested by enzyme-linked immunosorbent assay. Bone marrow cell cycle was performed by flow cytometry. HPLC was used to measure 20 potential chemical components related to myelosuppression, including ginsenoside Rg1, Re, Rb1, Rc, Rb2, Rb3, Rd, Rk3, Rh4, 20 (S)-Rg3, 20 (R)-Rg3, Rk1, Rg5, salidroside, and so on. ResultsG, LLA, and G-LLA improved the amount of peripheral blood cells and bone marrow cells of myelosuppressed mice (P < 0.01). They significantly increased the colony quantity of colony-forming unit–granulocyte macrophage, burst-forming unit−erythroid, colony-forming unit−erythroid, and colony-forming unit−megakaryocyte and amount of G2/M and S phase cells (P < 0.01). They also significantly decreased the amount of hematopoiesis-related cytokines (P < 0.01). The content of chemical components in G-LLA changed, and the change of rare saponin was the most obvious. ConclusionThese results show that G-LLA herb pair might produce synergistic or complementary compatibility effects on bone marrow suppression after chemotherapy. It suggests that the substance basis of G-LLA for treating bone marrow suppression may be effective chemical components.

“Function First” Screen of Primary AML Cells Identifies Common and Personalised Therapeutic Targets

BackgroundAcute myeloid leukaemia (AML) is a tremendously heterogeneous clonal disorder of haemopoietic progenitor cells and is the most common malignant myeloid disorder in adults. Identified mutations from genomic data cannot provide information about their therapeutic significance without functional data. Hereby, we applied a pooled shRNA library screen to identify the activated signalling pathways essential for the survival of AML cells.MethodsMononuclear cells from seven karyotypically normal AML patients were separated from peripheral blood or bone marrow aspirate at diagnosis and transduced with a pooled shRNA library containing 27500 shRNAs targeting 5000 individual genes (Human Module 1, Decipher, Cellecta). The targeted genes were components of known signalling pathways. At 72h post transduction, 30% of the cells were stored for baseline measurements and the rest were co-cultured with HS-5 stromal cells for the selection period. DNA was then extracted and the shRNA barcodes were sequenced on the Illumina NextSeq platform. The frequency of barcode appearance after the selection period was compared to their prevalence at baseline to calculate the shRNA depletion. The shRNAs were filtered to identify those genes which had multiple shRNA meeting a threshold depletion.ResultsOur data analysis of the 7 AML samples identified various signalling pathways for each of these patients. These data support the notion of heterogeneity in AML. The top 100 depleted genes (with depletion in at least 3 shRNA per gene) in each patient were selected and compared. Our limited initial data showed there to be several activated signalling pathways for each AML sample indicating that inhibition of more than one gene or pathway might be required for efficiently suppressing these leukaemia cells.Common targets: NOX1 was the most commonly identified therapeutic target among the screened patients being significantly depleted in AML cells from 5/7 patients. This is an important finding as there are available NOX1 inhibitors for treatment of colon cancers and can be investigated as a therapeutic option for acute myeloid leukaemia. The other most common targets were CDK5R1, DISC1, FSCN3, and PSMB7 which were found to be significantly depleted among 3 of the 7 screened patients. The merged data also showed 58 essential genes for AML cell survival were common in at least 2/7 patients. Using Enrichr the activated signalling pathways based on the top selected genes were identified. Various signalling pathways were observed for each patient showcasing the heterogeneity among AML patients (Figure 1). However, some signalling pathways were indeed common among multiple patients - with different genes being responsible for the activation of those pathways among the patients. The most common pathway was the metabolic pathway which was observed among the top 20 essential pathways in 6/7 patients. The JAK-STAT5 signalling pathway, purine metabolism and cAMP signalling pathway were also among the top 20 essential pathways in 3/7 patients while the following pathways: FoxO, PI3K-AKT, HIF-1, P53, Glucagon, and proteasome were observed in 2/7 patients. Identification of several essential survival pathways provides the opportunity to develop personalised therapy through combined targeting of more than one pathway.ConclusionThe signalling pathways analysis using candidate genes from a pooled shRNA library screen showed patient-specific signalling pathways and also common pathways among these screened patients. Absence of a common gene among the screened patients further highlights the significance of personalised therapy in AML and the necessity of developing diagnostic tools to identify potential targets at diagnosis. Identification of crucial genes such as NOX1 (a gene known to have a role in the survival of leukemic stem cells) and other genes with known significance in the pathogenesis of AML supports the application of this method for identifying therapeutic targets at diagnosis or relapse. [Display omitted] DisclosuresKnapper:Jazz: Other: Meeting and travel support; Daiichi Sankyo: Other: Meeting and travel support; Chroma Therapeutics: Research Funding; Celgene: Other: Meeting and travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Apperley:Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau.

Inverse and correlative relationships between TRIBBLES genes indicate non-redundant functions during normal and malignant hemopoiesis

TRIBBLES pseudokinases (TRIB1, TRIB2, and TRIB3) are important regulators of normal and malignant hemopoiesis. The relative abundance of each TRIBBLES family member may be important for distinct oncogenic or tumor suppressor functions. We map the expression profiles of TRIB1, TRIB2, and TRIB3 in human and murine hemopoietic stem, progenitor and mature cells, and in human leukemia datasets. Our data show that TRIB1-TRIB2 have an inverse expression relationship in normal hemopoiesis, whereas TRIB1-TRIB3 have a positive correlation. We reveal that TRIB3 expression is high in the dormant hemopoietic stem cell (HSC) population, implicating a novel role for TRIB3 in stem cell quiescence. These analyses support a non-redundant role for each TRIBBLES member during normal hemopoietic differentiation. We show that TRIB1-TRIB2 display a significant negative correlation in myelodysplastic syndrome and acute myeloid leukemia (AML) subtypes, but not in acute lymphoid leukemia. This inverse relationship is specific to certain subtypes of AML. A positive correlation exists in different leukemia subtypes between TRIB1-TRIB3. The TRIB1-TRIB2 and TRIB1-TRIB3 correlations are consistent with a correlative relationship with C/EBP transcription factor family members. Our results have implications for the development of strategies to therapeutically target these genes in different types of leukemia.

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD

BackgroundInterleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD.MethodsIL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non-smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry.ResultsIL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001).ConclusionsOur results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients.

Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2.

Trib2 pseudokinase has oncogenic and tumour suppressive functions depending on the cellular context. We investigated the ability of Trib2 to transform different haemopoietic stem and progenitor cells (HSPCs). Our study identified the granulocyte-macrophage progenitor (GMP) subpopulation as a potent leukaemia initiating cell of Trib2-driven AML in vivo. Trib2 transformed GMPs generated a fully penetrant and short latency AML. AML cells expressing elevated Trib2 led to a chemoresistant phenotype following chemotherapy treatment. We show that Trib2 overexpression results in an increase in BCL2 expression, and high Trib2 expressing cells are highly sensitive to cell killing by BCL2 inhibition (ABT199). Combined treatment with chemotherapeutic agents and BCL2 inhibition resulted in synergistic killing of Trib2+ AML cells. Trib2 transformed GMP AML cells showed more chemoresistance compared with HSPC derived Trib2 AML cells associated with higher Bcl2 expression. There is significant correlation of high TRIB2 and BCL2 expression in patient derived human AML cells. These data demonstrate that the cell of origin influences the leukaemic profile and chemotherapeutic response of Trib2+ AML. Combined TRIB2 and BCL2 expression in AML cells may have clinical utility relevant for monitoring drug resistance and disease relapse.

Anti-apoptotic A1 is not essential for lymphoma development in E\xb5-Myc mice but helps sustain transplanted E\xb5-Myc tumour cells

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-XL, but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a−/−A1-bfl/flA1-c−/− Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The human hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but remain incompletely characterized. Here, we demonstrated cord blood lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP), granulocyte-macrophage progenitor (GMP) and multi-lymphoid progenitor (MLP) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Though most progenitors had uni-lineage myeloid or lymphoid potential, bi- and rarer multi-lineage progenitors occurred in LMPP, GMP and MLP. This, coupled with single cell expression analyses, suggested a continuum of progenitors execute lymphoid and myeloid differentiation rather than only uni-lineage progenitors being present downstream of stem cells.

Immunohaematological complications in patients with sickle cell disease after haemopoietic progenitor cell transplantation: a prospective, single-centre, observational study

Haemopoietic progenitor cell (HPC) transplantation can cure sickle cell disease. Non-myeloablative conditioning typically results in donor-derived erythrocytes and stable mixed chimerism of recipient-derived and donor-derived leucocytes. Exposure to donor antigens from the HPC graft and new red cell antibodies induced by transfusion can lead to immunohaematological complications. We assessed the incidence of such complications among HPC transplant recipients with sickle cell disease. The study population was all patients with sickle cell disease enrolled before March 31, 2015, in the three clinical trials of non-myeloablative HPC transplantation at the National Institutes of Health. We assessed formation of new red cell antibodies after transplantation and red cell incompatibility between donors and recipients. 61 patients were enrolled, 42 were HLA matched and 19 were haploidentical. Nine (15%) had immunohaematological complications. Before HPC transplantation, three patients had antibodies incompatible with their donors. After HPC transplantation, new red cell antibodies were seen in six patients (11 alloantibodies and two autoantibodies), among whom three developed antibodies incompatible with donor or recipient red cells and three developed compatible antibodies. The clinical course of complications was highly variable, from no severe effects attributable to antibodies, to sustained reticulocytopenia, to near-fatal haemolysis. We found no significant correlation between immunohaematological complications and graft failure, graft rejection, or death. Clinical effects ranged from seemingly not clinically important to potentially fatal. In patients with sickle cell disease, donor and recipient red cell phenotypes should be carefully assessed before transplantation to minimise and manage the risk of immunohaematological complications. Intramural Research Program and National Institutes of Health.

Absence of Mcm6 mimics haemopoietic ageing and has implications for leukaemic transformation

Maintenance of genomic stability is critical for tumour suppression. Deregulation of the DNA replication machinery leads to accumulation of replication stress and increased mutational events often leading to oncogenesis. Replication stress occurs during normal cell ageing; aged cells acquire more background mutations and show inefficient DNA damage repair response (DDRR). Here, we aimed to investigate whether background mutations affect haemopoietic stem and progenitor cell (HSPC) functionality and leukaemic transformation. We generated an ageing-like haemopoietic model using CRISPR lentiviral vectors to knockout (KO) Mcm6, a key factor of the DNA replication machinery whose expression decreases with age. Murine HSPCs were transduced with CRISPR empty vector (EV) or carrying guide RNAs (gRNAs) targeting Mcm6. HSPC functionality was assessed in colony forming cell assays. KO cells formed fewer, but morphologically bigger and more compact colonies indicating a defect in self-renewal/stemness. Flow cytometry analysis revealed that KO cells expressed significantly lower levels of ter119 and c-Kit markers and higher levels of CD71 suggesting a block in erythropoiesis in vitro. To elucidate Mcm6 KO effects in vivo, transduced HSPCs were injected into lethally irradiated mice. Mice that received KO cells (KO chimeras) had lower levels of CD19 B cell marker and higher CD11b and Gr-1 myeloid markers in the peripheral blood. Bone marrow cells isolated from the KO chimeric mice accumulated replication stress evident by an increase in γH2AX, and showed poor DDRR supported by the lower expression of key DDR genes. Finally, using EV and KO transduced 32D cells, we assessed leukaemic transformation following MLL-AF9 (MA9) oncogene expression. 32D-KO-MA9+ cells exhibited a proliferative advantage and expressed lower levels of the c-kit progenitor marker compared to controls indicating that DNA replication stress as a result of Mcm6 deficiency changes the leukaemic phenotype. Collectively, our data indicates that replication stress alters both haemopoietic differentiation and leukaemic transformation, reminiscent of HSPC aged cells and the increased incidence of AML in the aged.

Toll-like receptor-induced expression of epithelial cytokine receptors on haemopoietic progenitors is altered in allergic asthma.

Haemopoietic progenitor cells (HPC) migrate to sites of allergic inflammation where, upon stimulation with epithelial cytokines, they produce Th2 cytokines and differentiate into mature eosinophils and basophils. They also express Toll-like receptors (TLR) involved in antimicrobial responses. The objective of this study was to compare TLR expression on peripheral blood HPC and TLR-induced responses, in particular changes in epithelial cytokine receptors, in healthy and asthmatic subjects at baseline and following allergen challenge. Ten healthy and 11 allergic asthmatic subjects were studied. HPC-enriched cell populations were stimulated with TLR-2, TLR-4 or TLR-9 ligands. TLR expression by circulating HPC and interleukin (IL)-25 (IL-17RB), IL-33 (ST2) and thymic stromal lymphopoietin receptor (TSLPR) expression after TLR ligation were examined by flow cytometry at baseline and, in asthmatics, following allergen challenge. The effects of dexamethasone (Dex) on TLR-induced responses were also assessed. Asthmatics had significantly lower circulating HPC expressing TLR-2 and TLR-9 with a similar trend for TLR-4. TLR-4 stimulation of HPC yielded higher numbers of TSLPR+ cells in asthmatics compared with healthy subjects. A similar trend was seen for TLR-9 ligation, an effect further augmented by allergen inhalation. Allergen challenge also enhanced TLR-induced ST2 expression on HPC. Treatment with Dex in vitro increased TLR-4-induced TSLPR expression but had no effect on other epithelial cytokine receptors. These data demonstrate an interaction between allergen and TLR ligand exposure in asthmatics. Allergen inhalation augments the TLR-induced inflammatory response by HPC, possibly leading to increased "in situ haemopoiesis" through up-regulation of TSLPR. These findings show that HPC may be a part of the pro-inflammatory cascade in pathogen-induced asthma exacerbation through their increased responsiveness to TLR stimulation.

The knowledge among student nurses regarding Umbilical Cord Stem Cell Banking

What could be more precious than gifting a born child a way to fight against haemopoietic, genetic or immune system diseases for the rest of his or her life? Or helping someone to fight against them with easily available sources. Once umbilical cord was considered as a waste product, but in this new era; it was found that umbilical cord blood is the richest source of haemopoietic stem and pluripotent progenitor cells. This creates the new way of storing them for the further use i.e. Stem cell banking. A study was formulated to assess the knowledge level about umbilical cord stem cell banking among student nurses, compare the knowledge level of student nurses of selected nursing colleges and to associate the knowledge score with selected demographic variables of student nurses from selected colleges. A quantitative research approachwas adopted and non-experimental descriptive research design was used. After getting the administrative permissionpurposive sampling was used to select 100 samplesfrom fourth year B. Sc nursing of selected nursing colleges for assessing the knowledge about umbilical cord stem cell banking.The study findings showed that, 76% of student nurses having moderate knowledge, 23% of student nurses having poor knowledge about umbilical cord stem cell banking. There was no significant association were found between the knowledge score and selected demographic variables. Now ongoing researches are about the uses of stem cell therapy in curing cardiac, genetic, hematologic and endocrine disorders. So, the storing of umbilical cord blood and stem cells at the time of birth will act like a total insurance for lot more disease in life.

Hemopoietic Progenitor Cells from the Bone Marrow of Serpinb3A-/- Mice Are Radioresistant

Introduction: Serpins are a superfamily of serine proteases that regulate proteolytic pathways by utilizing a conformational change to bind to and inhibit multiple peptidases. Currently 36 human serpins have been identified, but the roles of most are not fully understood (Silverman et al., J Biol Chem. 285(32):24299-305, 2010). A majority of serpins inhibit serine proteases but serpins that inhibit lysosomal cytosine peptidases, granzymes, serine protease and calpains. Previous studies have shown that serpins play a critical role in cell survival by blocking lysosomal induced necrosis injury. Serpinb3A (Antichymotrypsin) has been identified as an inhibitor of the serine peptidases, chymotrypsin and cathepsin G. We now report that hematopoietic progenitor cells in the bone marrow of Serpinb3A-/- mice are radioresistant.Materials & Methods: Serpinb3A-/- and control Balb/c mice were obtained from Charles River and were housed five per cage according to University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) regulations. All protocols were approved by the University of Pittsburgh IACUC. Veterinary care was provided by the Division of Laboratory Animal Research of the University of Pittsburgh. Whole bone marrow was obtained by flushing the marrow from the femurs of sacrificed Serpinb3A-/- and Balb/c mice. Single cell suspensions were irradiated to doses ranging from 0 to 8 Gy using a at 70 cGy/min using a Shepherd Mark 1 137Cs γ-ray source (J.L. Shepherd, San Fernando, CA, USA). The cells were plated in 0.8% methylcellulose containing hematopoietic growth supportive medium (StemCell Technology product #3134) at varying densities in quadruplicate in Linbro plates (Fisher Scientific, Pittsburgh, PA, USA) and incubated at 37°C and 5% CO2 for 9-11 days at which time the number of colony forming unit granulocyte-macrophage (CFU-GM) colonies of 50 cells or greater were counted. The data from 5 experiments was analyzed using linear quadratic and single-hit, multi-target models.Results: CFU-GM from Serpinb3A-/- mouse marrow showed an increase in radiation resistance with an increase in the shoulder of the irradiation survival curve compared to Balb/c marrow (ň = 4.9 ± 1.4 and 1.4 ± 0.1, p = 0.0451, respectively). There was no significant difference in the Do for CFU-GM of the two mouse strains (Do = 1.48 ± 0.19 Gy for Serapinb3A-/- mice and 1.56 ± 0.06 Gy for Balb/c mice, p = 0.0997.Conclusions: The data support a possible role for the loss of Serpinb3a in providing a cell survival advantage for hematopoietic progenitor cells exposed to lethal doses of ionizing irradiation. This would suggest that the Serpinb3a target protease helps repair the cellular injury induced by ionizing radiation. Alternatively, the germ line loss of Serpinb3a may up-regulate the expression of other clade B serpins known to protect cells from injury. Ongoing studies will help determine the nature of this protective effect against radiation-induced cell death. DisclosuresNo relevant conflicts of interest to declare.

Treatment of aplastic anaemia with lower-dose anti-thymocyte globulin produces similar response rates and survival as per standard dose anti-thymocyte globulin schedules.

Aplastic anaemia (AA) is a rare acquired bone marrow failure syndrome resulting from the immune-mediated destruction of haemopoietic stem cells. For adults in whom first-line haemopoietic progenitor cell transplantation is not feasible, combination anti-thymocyte globulin (ATGAM) plus cyclosporine A is standard therapy; however, there are minimal data available regarding the optimal ATGAM dosage in terms of efficacy and survival. Our institutions have historically used different dosing protocols of ATGAM in the treatment of AA. We aimed to review the outcome of AA patients treated with these protocols and compare them to the published literature. We conducted a retrospective study of 31 adults who received first-line ATGAM for AA and compared response rates and survival between cohorts who received standard (40 mg/kg/day D1-4) versus lower-dose (15 mg/kg/day D1-5) ATGAM schedules. There were similar rates of response (64 vs 71%, P = 1.0), relapse (33 vs 33%, P = 1.0), transformation (14 vs 24%, P = 0.66) or infection (43 vs 47%, P = 1.0), respectively, between standard and lower-dose cohorts. At a median follow up of 24 months, there was no statistical difference between standard and lower-dose cohorts in either event-free (42.2 vs 64.7%, P = 0.91) or overall survival (73.1 vs 88.2%, P = 0.75). Our experience suggests that lower-dose ATGAM at 15 mg/kg/day D1-5 as treatment of AA produces similar responses and outcomes as per standard-dose ATGAM schedules. Prospective trials comparing ATGAM dose schedules in AA are warranted.

Therapeutic apheresis

Apheresis is a procedure to separate and collect or remove a particular blood component. Apheresis may be used for collecting a therapeutic dose of component, remove the circulating amount of harmful component/substances, or collect a particular blood cell/precursor from a patient for reinfusion. Therapeutic plasma exchange (TPE) is a therapeutic procedure in which the plasma is removed and replaced with a replacement solution. It is used primarily for the removal of pathological immunoglobulin. Red cell exchange is a procedure in which the patient's red cells are removed and replaced with the donor red cells. It is usually used in the case of sickle cell disease to prevent stroke. Mononuclear cell collection is a procedure used primarily to collect peripheral blood haemopoietic progenitor cells (HPC) for autologous or allogeneic transplantation. As the most frequently performed procedures, they will be described in some detail in this article. Therapeutic leucapheresis and thrombocytapheresis are less frequently performed and will be mentioned briefly.

61 - Effect of Prolonged Transportation on Haemopoietic Progenitor Cells for CD34 Positive Selected Allogeneic Transplants in Paediatrics: Evaluation of Cell Viability and Transplant Outcomes

Introduction: Haematopoietic progenitor cells (HPC) from matched unrelated donors (MUD) frequently require transportation from their site of collection, often internationally. Due to geographical isolation, allogeneic translant recipients in Australia often receive cells from international donors with transit times in excess of 24 hours.

Therapeutic plasma exchange performed in tandem with hemodialysis without supplemental calcium in the apheresis circuit.

Therapeutic plasma exchange (TPE) and hemopoietic progenitor cell (HPC) collection are apheresis procedures that can safely be performed in tandem with hemodialysis. Despite the return of citrate-anticoagulated blood to the patient during HPC collection, it is not necessary to administer supplemental calcium during these procedures because the ionized calcium concentration is restored as the returning blood passes through the dialyzer. It is not known whether this applies to TPE, in which a mixture of blood and pharmaceutical albumin, an avid binder of plasma ionized calcium, is returned to the patient through the dialyzer. We report on three dialysis-dependent patients who required TPE and underwent tandem treatments without supplemental calcium in the apheresis circuit. Overall, ionized calcium fell 4-12% (P = 0.0.024) and patients reported no symptoms of hypocalcemic toxicity. Tandem hemodialysis/TPE can be performed without supplemental calcium in the apheresis circuit. J. Clin. Apheresis 32:154-157, 2017. © 2016 Wiley Periodicals, Inc.

Differentiation of haemopoietic stem cells and progenitor cells from human pluripotent stem cells

BackgroundInduced pluripotent stem cells hold great promise as an unlimited source of clinical grade immunologically matched haemopoietic stem cells (HSCs) for transplantation. However, there are currently no robust, reproducible differentiation protocols for the production of engraftable HSCs. We aimed to characterise the production of haemopoietic progenitor cells (HPCs) and HSCs during differentiation of human embryonic stem cells and induced pluripotent stem cells. MethodsWe used a serum-free, feeder-free, good manufacturing practice-compliant differentiation protocol established by the Novosang Consortium for the production of red blood cells for transfusion. Cell phenotype was assessed by flow cytometry for markers of HPCs and HSCs. Cell function was assessed in vitro by the colony forming units in culture (CFU-C) assay and in vivo with a long-term transplantation assay. FindingsAfter preliminary studies, the work focused on days 6, 8, and 10 of the differentiation protocol. In seven independent experiments, HPCs were reproducibly produced at high levels with over 90% of cells expressing CD43 by day 10. Almost all CD41+ cells coexpressed CD43 at all timepoints (day 10, 65% CD43+CD41+). CFU-Cs emerged in large numbers from day 6 and increased by day 10 (mean 1000 colonies per 105 cells plated [SD 478], six experiments). Cytospin and flow cytometry confirmed production of erythroid, granulocyte and macrophage, macrophage, and mixed colonies. The human embryonic HSC markers—vascular endothelial cadherin and CD45—were coexpressed at low levels (mean 3·6% [SD 2·5], seven experiments). No human engraftment was detected when cells were injected into immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. InterpretationThis differentiation protocol reproducibly gave a high yield of HPCs. However, there was no evidence of engraftable HSCs being produced. This finding could indicate inadequate extrinsic signalling during differentiation. Further work will therefore focus on co-culturing human pluripotent stem cells with human stromal cells in an attempt to mimic the embryonic HSC niche. FundingWellcome Trust.

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects

Human Hemopoietic Progenitor Cell Toll-like and Thymic Stromal Lymphopoietin Receptor Expression and Function in Allergic Asthmatic Subjects