In Vivo Therapeutic Potential of Mesenchymal Stromal Cells Depends on the Source and the Isolation Procedure

SummaryOver the last several years, mesenchymal stromal cells (MSCs) have been isolated from different tissues following a variety of different procedures. Here, we comparatively assess the ex vivo and in vivo properties of MSCs isolated from either adipose tissue or bone marrow by different purification protocols. After MSC transplantation into a mouse model of hindlimb ischemia, clinical and histological analysis revealed that bone marrow MSCs purified on adhesive substrates exerted the best therapeutic activity, preserving tissue viability and promoting formation of new arterioles without directly transdifferentiating into vascular cells. In keeping with these observations, these cells abundantly expressed cytokines involved in vessel maturation and cell retention. These findings indicate that the choice of MSC source and purification protocol is critical in determining the therapeutic potential of these cells and warrant the standardization of an optimal MSC isolation procedure in order to select the best conditions to move forward to more effective clinical experimentation

Harnessing the Power of CAR-NK Cells: A Promising Off-the-Shelf Therapeutic Strategy for CD38-Positive Malignancies

Background: CD38 is highly expressed on multiple myeloma (MM) cells and has been successfully targeted by different target therapy methods. This molecule is a critical prognostic marker in both diffuse large B-cell lymphoma and chronic lymphocytic leukemia.Objective: We have designed and generated an anti-CD38 CAR-NK cell applying NK 92 cell line. The approach has potential application as an off-the-shelf strategy for treatment of CD38 positive malignancies.Methods: A second generation of anti-CD38 CAR-NK cell was designed and generated, and their efficacy against CD38-positive cell lines was assessed in vitro. The PE-Annexin V and 7-AAD methods were used to determine the percentage of apoptotic target cells. Flow cytometry was used to measure IFN-γ, Perforin, and Granzyme-B production following intracellular staining. Using in silico analyses, the binding capacity and interaction interface were evaluated.Results: Using Lentivirus, cells were transduced with anti-CD38 construct and were expanded. The expression of anti-CD38 CAR on the surface of NK 92 cells was approximately 25%. As we expected from in silico analysis, our designed CD38-chimeric antigen receptor was bound appropriately to the CD38 protein. NK 92 cells that transduced with the CD38 chimeric antigen receptor, generated significantly more IFN-γ, perforin, and granzyme than Mock cells, and successfully lysed Daudi and Jurkat malignant cells in a CD38-dependent manner.Conclusion: The in vitro findings indicated that the anti-CD38 CAR-NK cells have the potential to be used as an off-the-shelf therapeutic strategy against CD38-positive malignancies. It is recommended that the present engineered NK cells undergo additional preclinical investigations before they can be considered for subsequent clinical trial studies

Sensory and Motor Behavior Evidences Supporting the Usefulness of Conditioned Medium from Dental Pulp-Derived Stem Cells in Spinal Cord Injury in Rats

Study Design Experimental animal study. Purpose This study aimed to assess effects of conditioned medium (CM) of dental pulp-derived stem cells loaded in collagen hydrogel on functional recovery following spinal cord injury (SCI). Overview of Literature SCI affects sensory and motor functions, and behavioral recovery is the most essential purpose of therapeutic intervention. Recent studies have reported that CM from dental pulp-derived stem cells has therapeutic benefits. In addition, collagen hydrogel acts as a drug delivery system in SCI experiments. Methods Stem cells from human exfoliated deciduous teeth (SHEDs) were cultured, and SHED-CM was harvested and concentrated. Collagen hydrogel containing SHED-CM was prepared. The rats were divided into five groups receiving laminectomy, compressive SCI with or without intraspinal injection of biomaterials (SHED-CM), and collagen hydrogel with or without SHED-CM. Basso, Beattie, and Bresnahan (BBB) scoring, inclined plane, cold allodynia, and beam walk tests were performed for 6 weeks to assess locomotor, motor, sensory, and sensory-motor performances, respectively. Results Scores of the rats receiving SHED-CM loaded in collagen hydrogel were significantly better than those of the other injured groups at 1-week post-injury for BBB, 2 weeks for inclined plane, 2 weeks for cold allodynia, and 4 weeks for beam walk tests (p <0.05). The differences remained significant throughout the study. Conclusions Intraspinal administration of SHED-CM loaded in collagen hydrogel leads to improved functional recovery and proposes a cell-free therapeutic approach for SCI

Decellularized human ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated protocol, as a natural three-dimensional scaffold for construction of bioengineered ovaries

Abstract Background The increasing number of patients with ovarian insufficiency due to autoimmune disorders, genetic predisposition, or iatrogenic effects of treatment such as cancer therapies necessitates an urgent measure to find a safe and transplantable alternative ovary. A bioengineered ovary is one of the strategies on which the researchers have recently been working. An engineered ovary should be able to mimic the natural ovary aspects. Recent studies suggest that the decellularized organ-specific extracellular matrix-based scaffolds can serve as a native niche to bioengineering artificial organs. Therefore, we established a human decellularized ovarian scaffold based on a sodium lauryl ester sulfate (SLES)-treated process, as an optimized protocol. Methods The human ovary samples were decellularized with 1% SLES for 48 h followed by DNase I in PBS for 24 h, and then thoroughly rinsed in PBS to remove the cell remnants and chemical reagents. Efficient cell removal was confirmed by DNA content analysis, hematoxylin and eosin, and Hoechst staining. Preservation assessment of the extracellular matrix structures was performed by immunohistochemistry, histological staining, and scanning electron microscopy. An MTT test was done to assess the in vitro scaffold’s cytocompatibility, and finally in vivo studies were performed to evaluate the biocompatibility, bioactivity, and secretion functions of the ovarian grafts made of primary ovarian cells (POCs) on the decellularized scaffolds. Results Evidence provided by SEM, histochemical, and immunohistochemical analyses showed that the ovarian extracellular matrix was preserved after decellularization. Moreover, MTT test indicated the suitable cytocompatibility of the scaffolds. The in vivo assessment showed that the POCs kept their viability and bioactivity, and reconstructed the primordial or primary follicle-like structures within the scaffolds after transplantation. Immunostaining characterized somatic cells that were capable of expressing steroid hormone receptors; also, as a marker of granulosa cell, inhibin-α immunostaining demonstrated these cells within the grafts. Additionally, hormone assessment showed that serum estradiol and progesterone levels were significantly higher in ovariectomized rats with ovarian cells-seeded grafts than those with or without decellularized scaffold grafts. Conclusions A human ovary-specific scaffold based on a SLES-decellularized protocol as a biomimicry of the natural ovarian niche can be an ideal scaffold used to reconstruct the ovary

Comparing the effects of fetal bovine and human sera on isolation, expansion and osteogenic differentiation of adipose-derived stem cells

Background & Objectives: Adipose-derived stem cells (ADSCs) have been investigated as a promising cell source for therapeutic and engineering applications. Common proliferation protocols use fetal bovine serum (FBS) as a growth factor which is potentially a source of pathogens, and contains animal antigens that can cause an allergic reaction after transplantation in the recipient, and regarding immunity, human serum (HS) could be a suitable alternative to it. The aim of this study is to compare the culture medium enriched with FBS or HS as supplements in the proliferation and differentiation of ADSCs. Materials & Methods: Human serum was extracted from 90 ml venous blood of a healthy person, taken in respective intervals. The ADSCs were isolated according to the protocol from adipose tissue after lipolysis operation and cultured by mediums enriched with FBS or HS. Expression of the surface markers of ADSCs was investigated by flow cytometry. Osteogenic differentiation was evaluated by Alizarin Red staining method. Results: The analysis of cell growth showed that the isolation and proliferation of ADSCs in both media (HS & FBS) were similar, but there was a significant difference in case of differentiation in the HS medium. Conclusion: This study showed that FBS could be replaced by HS in case of isolation, proliferation and differentiation of stem cells studies as supplement. Furthermore, our data suggest a fast and safe proliferation protocol by using human serum in the stem cells culture and cell-based therapies. &nbsp

Deep analysis of N-cadherin/ADH-1 interaction: a computational survey

<p>Due to the considerable role of N-cadherin in cancer metastasis, tumor growth, and progression, inhibition of this protein has been highly regarded in recent years. Although ADH-1 has been known as an appropriate inhibitor of N-cadherin in clinical trials, its chemical nature and binding mode with N-cadherin have not been precisely specified yet. Accordingly, in this study, quantum mechanics calculations were used to investigate the chemical nature of ADH-1. These calculations clarify the molecular properties of ADH-1 and determine its reactive sites. Based on the results, the oxygen atoms are suitable for electrophilic reactivity, while the hydrogen atoms that are connected to nitrogen atoms are the favorite sites for nucleophilic reactivity. The higher electronegativity of the oxygen atoms makes them the most reactive portions in this molecule. Molecular docking and molecular dynamics (MD) simulation have also been applied to specify the binding mode of ADH-1 with N-cadherin and determine the important residues of N-cadherin involving in the interaction with ADH-1. Moreover, the verified model by MD simulation has been studied to extract the free energy value and find driving forces. These calculations and molecular electrostatic potential map of ADH-1 indicated that hydrophobic and electrostatic interactions are almost equally involved in the implantation of ADH-1 in the N-cadherin binding site. The presented results not only enable a closer examination of N-cadherin in complex with ADH-1 molecule, but also are very beneficial in designing new inhibitors for N-cadherin and can help to save time and cost in this field.</p

Plasticity comparison of two stem cell sources with different Hox gene expression profiles in response to cobalt chloride treatment during chondrogenic differentiation

The limited self-repair capacity of articular cartilage is a challenge for healing injuries. While mesenchymal stem/stromal cells (MSCs) are a promising approach for tissue regeneration, the criteria for selecting a suitable cell source remain undefined. To propose a molecular criterion, dental pulp stem cells (DPSCs) with a Hox-negative expression pattern and bone marrow mesenchymal stromal cells (BMSCs), which actively express Hox genes, were differentiated towards chondrocytes in 3D pellets, employing a two-step protocol. The MSCs’ response to preconditioning by cobalt chloride (CoCl2), a hypoxia-mimicking agent, was explored in an assessment of the chondrogenic differentiation’s efficiency using morphological, histochemical, immunohistochemical, and biochemical experiments. The preconditioned DPSC pellets exhibited significantly elevated levels of collagen II and glycosaminoglycans (GAGs) and reduced levels of the hypertrophic marker collagen X. No significant effect on GAGs production was observed in the preconditioned BMSC pellets, but collagen II and collagen X levels were elevated. While preconditioning did not modify the ALP specific activity in either cell type, it was notably lower in the DPSCs differentiated pellets compared to their BMSCs counterparts. These results could be interpreted as demonstrating the higher plasticity of DPSCs compared to BMSCs, suggesting the contribution of their unique molecular characteristics, including their negative Hox expression pattern, to promote a chondrogenic differentiation potential. Consequently, DPSCs could be considered compelling candidates for future cartilage cell therapy.<br/