Evaluation of a clinical-grade, cryopreserved, ex vivo-expanded stem cell product from cryopreserved primary umbilical cord blood demonstrates multilineage hematopoietic engraftment in mouse xenografts.

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). METHODS: cGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. RESULTS: The authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. CONCLUSIONS: The authors' results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.

A Parsimonious Host Inflammatory Biomarker Signature Predicts Incident Tuberculosis and Mortality in Advanced Human Immunodeficiency Virus.

BACKGROUND: People with advanced human immunodeficiency virus (HIV) (CD4 < 50) remain at high risk of tuberculosis (TB) or death despite the initiation of antiretroviral therapy (ART). We aimed to identify immunological profiles that were most predictive of incident TB disease and death. METHODS: The REMEMBER randomized clinical trial enrolled 850 participants with HIV (CD4 < 50 cells/µL) at ART initiation to receive either empiric TB treatment or isoniazid preventive therapy (IPT). A case-cohort study (n = 257) stratified by country and treatment arm was performed. Cases were defined as incident TB or all-cause death within 48 weeks after ART initiation. Using multiplexed immunoassay panels and ELISA, 26 biomarkers were assessed in plasma. RESULTS: In total, 52 (6.1%) of 850 participants developed TB; 47 (5.5%) died (13 of whom had antecedent TB). Biomarkers associated with incident TB overlapped with those associated with death (interleukin [IL]-1ß, IL-6). Biomarker levels declined over time in individuals with incident TB while remaining persistently elevated in those who died. Dividing the cohort into development and validation sets, the final model of 6 biomarkers (CXCL10, IL-1ß, IL-10, sCD14, tumor necrosis factor [TNF]-α, and TNF-ß) achieved a sensitivity of 0.90 (95% confidence interval [CI]: .87-.94) and a specificity of 0.71(95% CI: .68-.75) with an area under the curve (AUC) of 0.81 (95% CI: .78-.83) for incident TB. CONCLUSION: Among people with advanced HIV, a parsimonious inflammatory biomarker signature predicted those at highest risk for developing TB despite initiation of ART and TB preventive therapies. The signature may be a promising stratification tool to select patients who may benefit from increased monitoring and novel interventions. CLINICAL TRIALS REGISTRATION: NCT01380080.

Pre-clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice.

BACKGROUND: Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf-life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third-party, cryopreservable source of PLT-generating cells has the potential to complement presently available PLT transfusion products. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. RESULTS: We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune-deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. CONCLUSION: This is a proof-of-principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.

Ex Vivo Expansion of Adult Hematopoietic Stem and Progenitor Cells with Valproic Acid.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.