Reduced Osteogenic Differentiation Potential In Vivo in Acute Myeloid Leukaemia Patients Correlates with Decreased BMP4 Expression in Mesenchymal Stromal Cells.

The osteogenic differentiation potential of mesenchymal stromal cells (hMSCs) is an essential process for the haematopoiesis and the maintenance of haematopoietic stem cells (HSCs). Therefore, the aim of this work was to evaluate this potential in hMSCs from AML patients (hMSCs-AML) and whether it is associated with BMP4 expression. The results showed that bone formation potential in vivo was reduced in hMSCs-AML compared to hMSCs from healthy donors (hMSCs-HD). Moreover, the fact that hMSCs-AML were not able to develop supportive haematopoietic cells or to differentiate into osteocytes suggests possible changes in the bone marrow microenvironment. Furthermore, the expression of BMP4 was decreased, indicating a lack of gene expression committed to the osteogenic lineage. Overall, these alterations could be associated with changes in the maintenance of HSCs, the leukaemic transformation process and the development of AML.

Extracellular Matrix-Modified Fiber Scaffolds as a Proadipogenic Mesenchymal Stromal Cell Delivery Platform.

Melt electrowriting (MEW) is an additive manufacturing technology that produces readily handleable fibrous scaffolds with controlled geometry to support cell infiltration. Although MEW scaffolds have excellent potential for cell delivery in regenerative medicine applications, studies to date have primarily focused on polymers such as poly(ε-caprolactone) (PCL) that lack bioactive cues to affect cell function. To address this aspect, MEW scaffolds with extracellular matrix (ECM) coatings were developed as a proadipogenic platform for human mesenchymal stromal cells (hMSCs). More specifically, highly flexible PCL scaffolds fabricated through MEW were coated with a complex ECM suspension prepared from human decellularized adipose tissue (DAT), purified fibronectin, or laminin to determine the effects of two key bioactive proteins present within adipose-derived ECM. In vitro studies exploring the response of human bone marrow-derived mesenchymal stromal cells cultured under adipogenic differentiation conditions indicated a high level of differentiation on all substrates studied, including unmodified PCL scaffolds and two-dimensional controls. To more fully assess the intrinsic proadipogenic capacity of the composite biomaterials, a modified culture regime was established that involved a short-term adipogenic induction in differentiation medium, followed by continued culture in maintenance medium supplemented with insulin for up to 3 weeks. Under these conditions, adipogenic differentiation was enhanced on all fiber scaffolds as compared to the tissue culture controls. Notably, the highest adipogenic response was consistently observed on the PCL + DAT scaffolds, based on the analysis of multiple markers including adipogenic gene [lipoprotein lipase, fatty acid binding protein 4 (FABP4), adiponectin, perilipin 1] and protein (FABP4, leptin) expression and intracellular triglyceride accumulation. Taken together, the PCL scaffolds incorporating DAT provide an adipoinductive microenvironment for the hMSCs, with particular applicability of this cell-instructive delivery platform for applications in plastic and reconstructive surgery.

Donor Variability in Growth Kinetics of Healthy hMSCs Using Manual Processing: Considerations for Manufacture of Cell Therapies.

Human mesenchymal stromal cells (hMSCs) are excellent candidates for cell therapy but their expansion to desired clinical quantities can be compromised by ex vivo processing, due to differences between donor material and process variation. The aim of this article is to characterize growth kinetics of healthy baseline "reference" hMSCs using typical manual processing. Bone-marrow derived hMSCs from ten donors are isolated based on plastic adherence, expanded, and analyzed for their growth kinetics until passage 4. Results indicate that hMSC density decreases with overall time in culture (p < 0.001) but no significant differences are observed between successive passages after passage 1. In addition, fold increase in cell number dropped between passage 1 and 2 for three batches, which correlated to lower performance in total fold increase and expansion potential of these batches, suggesting that proliferative ability of hMSCs can be predicted at an early stage. An indicative bounded operating window is determined between passage 1 and 3 (PDL < 10), despite the high inter-donor variability present under standardized hMSC expansion conditions used. hMSC growth profile analysis will be of benefit to cell therapy manufacturing as a tool to predict culture performance and attainment of clinically-relevant yields, therefore stratifying the patient population based on early observation.

The molecular signature of AML mesenchymal stromal cells reveals candidate genes related to the leukemogenic process.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by myeloid precursor proliferation in the bone marrow, apoptosis reduction and differentiation arrest. Although there are several studies in this field, events related to disease initiation and progression remain unknown. The malignant transformation of hematopoietic stem cells (HSC) is thought to generate leukemic stem cells, and this transformation could be related to changes in mesenchymal stromal cell (hMSC) signaling. Thus, the aim of this work was to analyze the gene expression profile of hMSC from AML patients (hMSC-AML) compared to healthy donors hMSCs (hMSC-HD). The results showed a common molecular signature for all hMSC-AML. Other assays were performed with a large number of patients and the results confirmed a molecular signature that is capable of distinguishing hMSC-AML from hMSC-HD. Moreover, CCL2 and BMP4 genes encode secreted proteins that could affect HSCs. To verify whether these proteins are differentially expressed in AML patients, ELISA was performed with plasma samples. CCL2 and BMP4 proteins are differentially expressed in AML patients, indicating changes in hMSC-AML signaling. Altogether, hMSCs-AML signaling alterations could be an important factor in the leukemic transformation process.