The proteomic and particle composition of human platelet lysate for cell therapy products.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications.

An Experimental Study Comparing the Expansion of Peripheral Blood Natural Killer (NK) Cells Cultured with Artificial Antigen-Presenting Cells, in the Presence or Absence of Bone Marrow Mesenchymal Stem Cells (MSCs).

NK cells have been seen as potential agents in adoptive immunotherapy for cancer. The main challenge for the success of this approach is to obtain a great quantity of activated NK cells for adoptive transfer. The present study had aimed to evaluate the effect of a feeder layer of irradiated MSCs in the in vitro expansion of NK cells. MSCs were obtained from the bone marrow (BM) cells remaining in the bag and filter used in the transplantation of hematopoietic stem cells. NK cells were obtained from peripheral blood (PB) of healthy volunteers. NK expansion and activation were stimulated by culture with artificial antigen-presenting cells (aAPCs) and IL-2, in the presence or absence of BM-MSCs. NK cell proliferation, phenotypic expression and cytotoxic activity were evaluated. Both culture conditions showed high NK purity with predominance of NK CD56brightCD16+ subset post expansion. However, cultures without the presence of MSCs showed higher NK proliferation, expression of activation markers (CD16 and NKG2D) and related cytotoxic activity. In this experimental study, the presence of a feeder layer of irradiated BM-MSCs interfered negatively in the expansion of PB-NKs, limiting their growth and activation. Further investigation is needed to understand the mechanisms of NK-MSC interaction and its implications.

Donor characteristics and hematopoietic stem cell transplantation outcome: experience of a single center in Southern Brazil.

BACKGROUND: Hematopoietic stem cell transplantation is a curative treatment for many patients with hematological disorders. Donor-recipient genetic disparity, especially involving the human leukocyte antigen system is a critical factor for transplant outcome. OBJECTIVE: To evaluate retrospectively donor characteristics and correlations with the occurrence of acute and chronic graft-versus-host disease, disease-free survival and overall survival in a Brazilian population submitted to allogeneic hematopoietic stem cell transplantation between 1994 and 2012 in a single center. RESULTS: Three hundred and forty-seven consecutive transplantations were included. Related transplants (81.2%) were significantly more common than unrelated transplants (18.7%); donor and recipient median ages were 34 (range: 1-61) and 33 (range: 3-65) years respectively with donor HLAs being matched for 333 (95.9%) patients. Donor gender, cytomegalovirus status and ABO incompatibility did not influence the five-year overall survival. In univariate analyses, overall survival was negatively influenced by the presence of acute graft-versus-host disease (33% vs. 47%, respectively; p-value = 0.04), unrelated transplant (41.5% vs. 50.9%, respectively; p-value = 0.045) and donors aged over 40 years (41% vs. 52%, respectively; p-value = 0.03). Older donors were associated with a higher rate of acute (52% vs. 65.8%; p-value = 0.03) and chronic graft-versus-host disease (60% vs. 43%, respectively; p-value = 0.015). In multivariate analyses, acute graft-versus-host disease [relative risk (RR): 1.8; 95% confidence interval (CI): 1.1-29; p-value = 0.008] and older donors (RR: 1.6; 95% CI 1.11-2.24; p-value = 0.013) were associated with higher transplant-related mortality. CONCLUSIONS: In transplant patients, to have a donor older than 40 years of age seems to significantly increase the incidence of acute and chronic graft-versus-host disease and transplant-related mortality with no impact on disease-free survival and overall survival. In spite of the rather small cohort of patients, these findings are similar to what is described in the literature suggesting that a younger donor should be chosen whenever possible.

Donor characteristics and hematopoietic stem cell transplantation outcome: experience of a single center in Southern Brazil

ABSTRACT Background: Hematopoietic stem cell transplantation is a curative treatment for many patients with hematological disorders. Donor-recipient genetic disparity, especially involving the human leukocyte antigen system is a critical factor for transplant outcome. Objective: To evaluate retrospectively donor characteristics and correlations with the occurrence of acute and chronic graft-versus-host disease, disease-free survival and overall survival in a Brazilian population submitted to allogeneic hematopoietic stem cell transplantation between 1994 and 2012 in a single center. Results: Three hundred and forty-seven consecutive transplantations were included. Related transplants (81.2%) were significantly more common than unrelated transplants (18.7%); donor and recipient median ages were 34 (range: 1-61) and 33 (range: 3-65) years respectively with donor HLAs being matched for 333 (95.9%) patients. Donor gender, cytomegalovirus status and ABO incompatibility did not influence the five-year overall survival. In univariate analyses, overall survival was negatively influenced by the presence of acute graft-versus-host disease (33% vs. 47%, respectively; p-value = 0.04), unrelated transplant (41.5% vs. 50.9%, respectively; p-value = 0.045) and donors aged over 40 years (41% vs. 52%, respectively; p-value = 0.03). Older donors were associated with a higher rate of acute (52% vs. 65.8%; p-value = 0.03) and chronic graft-versus-host disease (60% vs. 43%, respectively; p-value = 0.015). In multivariate analyses, acute graft-versus-host disease [relative risk (RR): 1.8; 95% confidence interval (CI): 1.1-29; p-value = 0.008] and older donors (RR: 1.6; 95% CI 1.11-2.24; p-value = 0.013) were associated with higher transplant-related mortality. Conclusions: In transplant patients, to have a donor older than 40 years of age seems to significantly increase the incidence of acute and chronic graft-versus-host disease and transplant-related mortality with no impact on disease-free survival and overall survival. In spite of the rather small cohort of patients, these findings are similar to what is described in the literature suggesting that a younger donor should be chosen whenever possible.

Effects Of Hypoxia in Long-Term In Vitro Expansion of Human Bone Marrow Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are considered multipotent stromal, non-hematopoietic cells with properties of self-renovation and differentiation. Optimal conditions for culture of MSC have been under investigation. The oxygen tension used for cultivation has been studied and appears to play an important role in biological behavior of mesenchymal cells. The aim is characterize MSC in hypoxia and normoxia conditions comparing their morphological and functional characteristics. Bone marrow-derived mesenchymal stem cells obtained from 15 healthy donors and cultured. MSC obtained from each donor were separated into two cultivation conditions normoxia (21% O2 ) and hypoxia (three donors at 1%, three donors at 2%, five donors at 3%, and four donors at 4% O2 ) up to second passage. MSC were evaluated for proliferation, differentiation, immunophenotyping, size and cell complexity, oxidative stress, mitochondrial activity, and autophagy. Culture conditions applied did not seem to affect immunophenotypic features and cellular plasticity. However, cells subjected to hypoxia showed smaller size and greater cellular complexity, besides lower proliferation (P < 0.002). Furthermore, cells cultured in low O2 tension had lower mitochondrial activity (P < 0.03) and a reduced tendency to autophagy, although oxidative stress did not vary among groups (P < 0.39). Oxygen tension seems to be a key regulator of cellular adaptation in vitro, and metabolic effects underlying this variable remain undescribed. Heterogeneity or even lack of results on the impact of oxygen concentration used for expanding MSC highlights the need for further research, in order to optimize conditions of cultivation and expansion and achieve greater safety and therapeutic efficacy. J. Cell. Biochem. 118: 3072-3079, 2017. © 2017 Wiley Periodicals, Inc.