A phase 1/2 study of the combination of acalabrutinib and vistusertib in patients with relapsed/refractory B-cell malignancies.

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

Adipose Stem Cells Incorporated in Fibrin Clot Modulate Expression of Growth Factors.

PURPOSE: To evaluate the platelet capture rate of whole blood fibrin clots and the expression, secretion, and retention of the growth factors vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) from fibrin clots and to determine how these levels may be modulated by allogeneic adipose-derived stem cells (ASCs). METHODS: Whole blood from 10 human volunteers was transferred to a clotting device and the platelet capture rate determined. Two experimental conditions and 1 control were evaluated over 2 weeks in vitro. Clots made from human whole blood without ASCs, clot(-)ASC, were compared with clots with ASCs incorporated, clot(+)ASC, and a control group of synthetic polyethylene glycol gels with ASCs incorporated, control(+)ASCs. All conditions were examined for secretion and retention of VEGF, PDGF, and bFGF via enzyme-linked immunosorbent assay and immunohistochemistry. The analysis of platelet retention for clots made with this device was performed. RESULTS: Enzyme-linked immunosorbent assay analysis showed significantly higher (P < .001) secretion of VEGF in clot(+)ASC compared with clot(-)ASC or control(+)ASC. In contrast, clot(-)ASC produced soluble PDGF, and the addition of ASCs results in decreased soluble PDGF with concomitant increases in PDGF immunoreactivity of ASCs. Soluble bFGF levels were low in clot(-)ASC, and were found to increase at early time points in clot(+)ASC. Furthermore, bFGF immunoreactivity could be detected in clot(+)ASC, whereas no bFGF immunoreactivity is present in clot(-)ASC or control(+)ASC. Control(+)ASC displayed a spike in bFGF secretion at day 0, which may be due to a stress response elicited by the encapsulation process. Approximately 98% of available platelets in whole blood were concentrated in the clot on formation. CONCLUSIONS: Fibrin clots made by this method retain high concentrations of platelets, and when incorporated with ASCs show modulated secretion and immunoreactivity of VEGF, PDGF, and bFGF. CLINICAL RELEVANCE: Whole blood fibrin clots capture platelets and release growth factors, and the addition of ASCs increases VEGF release for up to 2 weeks after clot formation. This suggests that whole blood fibrin clots may be a viable scaffold and delivery vehicle for future stem cell treatments.

Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells.

One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg-Gly-Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg-Gly-Asp-matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration.

Strategies for bioengineered scaffolds that support adipose stem cells in regenerative therapies.

Regenerative medicine possesses the potential to ameliorate damage to tissue that results from a vast range of conditions, including traumatic injury, tumor resection and inherited tissue defects. Adult stem cells, while more limited in their potential than pluripotent stem cells, are still capable of differentiating into numerous lineages and provide feasible allogeneic and autologous treatment options for many conditions. Adipose stem cells are one of the most abundant types of stem cell in the adult human. Here, we review recent advances in the development of synthetic scaffolding systems used in concert with adipose stem cells and assess their potential use for clinical applications.

Induced Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium: A Comparative Study Between Cell Lines and Differentiation Methods.

PURPOSE: The application of induced pluripotent stem cell-derived retinal pigmented epithelium (iPSC-RPE) in patients with retinal degenerative disease is making headway toward the clinic, with clinical trials already underway. Multiple groups have developed methods for RPE differentiation from pluripotent cells, but previous studies have shown variability in iPSC propensity to differentiate into RPE. METHODS: This study provides a comparison between 2 different methods for RPE differentiation: (1) a commonly used spontaneous continuously adherent culture (SCAC) protocol and (2) a more rapid, directed differentiation using growth factors. Integration-free iPSC lines were differentiated to RPE, which were characterized with respect to global gene expression, expression of RPE markers, and cellular function. RESULTS: We found that all 5 iPSC lines (iPSC-1, iPSC-2, iPSC-3, iPSC-4, and iPSC-12) generated RPE using the directed differentiation protocol; however, 2 of the 5 iPSC lines (iPSC-4 and iPSC-12) did not yield RPE using the SCAC method. Both methods can yield bona fide RPE that expresses signature RPE genes and carry out RPE functions, and are similar, but not identical to fetal RPE. No differences between methods were detected in transcript levels, protein localization, or functional analyses between iPSC-1-RPE, iPSC-2-RPE, and iPSC-3-RPE. Directed iPSC-3-RPE showed enhanced transcript levels of RPE65 compared to directed iPSC-2-RPE and increased BEST1 expression and pigment epithelium-derived factor (PEDF) secretion compared to directed iPSC-1-RPE. In addition, SCAC iPSC-3-RPE secreted more PEDF than SCAC iPSC-1-RPE. CONCLUSIONS: The directed protocol is a more reliable method for differentiating RPE from various pluripotent sources and some iPSC lines are more amenable to RPE differentiation.

Vitronectin-Based, Biomimetic Encapsulating Hydrogel Scaffolds Support Adipogenesis of Adipose Stem Cells.

Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVß5 and α1ß1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects.