Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton's jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Scaffolds, both natural and synthetic, used in tissue engineering provide mechanical support to cells. Tissue decellularization has been used to provide natural extracellular matrix scaffolds for tissue engineering purposes. In this chapter we focus on describing the methodology used to decellularize Wharton's jelly matrix, the mucous connective tissue that surrounds umbilical cord vessels, to obtain decellularized Wharton's jelly matrix (DWJM); an extracellular matrix that can be used for tissue engineering purposes. We also, briefly, describe our experience with processing DWJM for cell seeding and recellularization.

Background: Previously, we demonstrated that host pre-treatment with hyperbaric oxygen (HBO) improves engraftment of umbilical cord blood (UCB) CD34+ cells in a transplant murine model. Recent data suggest that erythropoietin (EPO) determines the fate of hematopoietic progenitor cells in favor of erythroid differentiation. Herein, we evaluated mechanisms underlying the favorable effect of HBO therapy on UCB CD34+ cell engraftment. We hypothesized that HBO modulates which in turn enhances UCB CD34 homing to the marrow, thereby enhancing engraftment. Materials/methods: We examined receptor (EPOR) expression on UCB CD34+ cells by flow cytometry and western blot. Transmigration assays were used to evaluate effects on UCB CD34+ cell migration toward Stromal Cell-Derived Factor 1 (SDF-1; CXCL12) which has been shown to mediate CD34+ chemotaxis to the marrow. Using an NSG transplant murine model, we evaluated serum levels by ELISA and early UCB bone marrow/spleen homing by flow cytometry. Bone marrow UCB cell differentiation was examined 1 and 2 weeks post-transplant using CFU-assay. Results: We found that on average 17.6 (4.9-38.7) % of CD34+ enriched UCB cells express EPOR. EPOR expression was confirmed by western blot. The percentage of EPOR positive cells was significantly higher in hematopoietic stem cells (HSC) defined as Lin- CD34+ CD38- CD45RA- CD90+ CD49f+ (45.7+/-1.4) compared to hematopoietic progenitor cells defined as Lin- CD34+ CD38+ (25.1+/-0.7) and multipotent progenitors defined as Lin- CD34+ CD38- CD45RA- CD90- CD49f- (27.2+/-0.3). Also, exposure of UCB CD34+ cells to during cell culture inhibited their migration toward SDF-1 (Figure-1). HBO treated mice demonstrated significantly lower serum blood levels compared to control mice (727.4+/- 42.02 versus1576 +/- 80.90 pg/mL, p u003c0.0001) measured 3-hours post-transplant. A higher percentage of human CD34+ was seen in the bone marrow of HBO treated mice 3-hours post-transplant (6.8+/-1.0 versus 3.4+/-0.6), p=0.01). HBO treated mice demonstrated significantly lower numbers of BFU-E ( 26.7+/-8.7 versus 75.3+/-19.3, p =0.043) and increasing numbers of CFU-G/M (166.0+/-27.9 versus 74.8+/-32.6, p =0.05). Conclusions: EPOR is enriched in UCB HSC. HBO lowers host blood and as a result improves UCB CD34+ cell early bone marrow homing and enhances myeloid differentiation.![Figure 1.][1] Figure 1. EPO effects on UCB CD34+ cell transmigration toward SDF-1 gradient.Disclosures No relevant conflicts of interest to declare. [1]: pending:yes

Background and objective: Acute myeloid leukemia (AML) develops from leukemia stem cells (LSCs), a small subset of leukemia cells possessing both self-renewal and multilineage differentiation potential similar to normal hematopoietic stem cells. The stem cell niche of the bone marrow microenvironment protects LSCs from chemotherapy, resulting in subsequent leukemia relapse. The study of AML LSCs in vitro is limited because of the lack of an ideal culture system mimicking the protective bone marrow microenvironment. The bone marrow stem cell niche is mainly composed of stromal cells, soluble cytokines and growth factors, as well as extracellular matrix (ECM). We therefore developed a 3-dimensional ECM model using decellularized Wharton's jelly from human umbilical cords to better characterize AML LSCs in vitro. Previously we have shown that leukemia cells grown in DWJM changed morphology to become spindle shaped and maintained viability but had decreased proliferation as measured by Alamar blue assay. Herein, we further characterize leukemia cells cultured in DWJM.

The disparate response of leukemia cells to chemotherapy in vivo, compared to in vitro, is partly related to the interaction of leukemic cells and the three-dimensional bone marrow stromal microenvironment. We investigated the effects of chemotherapy agents on leukemic cell lines co-cultured with human bone marrow mesenchymal stem cells (hu-BM-MSCs) in a three-dimensional model (3D). Comparison was made to leukemic cells treated in suspension, or grown on a hu-BM-MSC monolayer (2D conditions). We demonstrated that leukemic cells cultured in 3D were more resistant to drug-induced apoptosis compared to cells cultured in 2D or in suspension. We also demonstrated significant differences in leukemic cell response to chemotherapy using different leukemic cell lines cultured in 3D. We suggest that the differential responses to chemotherapy in 3D may be related to the expression of N-cadherin in the co-culture system. This unique model provides an opportunity to study leukemic cell responses to chemotherapy in 3D.

Umbilical cord blood (UCB) is of great value by providing transplantable hematopoietic stem and progenitor cells (HSPCs). Compared with HSPCs from adult bone marrow and periferial blood, UCB cells are more primitive with higher proliferation ability, and UCB HSPC transplantation requires less HLA matching. The major problems in UCB transplantation is the limited number of transplantable cells in each unit which are often insufficient for transplantation in adults

Abstract Abstract 4882 Introduction: Current in vitro drug testing models are based on 2-dimensional (2D) cell culture systems and therefore do not always predict in vivo responses. This lack of predictability of the 2D assays is believed to be related to the 3-dimensional (3D) microenvironment present in tissues or tumors. This 3D microenvironment, were cell-cell and cell-extracellular matrix (ECM) interactions occur, is fundamental for cell biologic activities. This is especially true for acute myeloid leukemia, were current 2-D cell culture models do not always predict clinical responses. This discrepancy in leukemia cell responses to chemotherapy in vivo, in comparison to in vitro, is at least partly related to leukemia cells interaction with the bone marrow microenvironment and their ability to establish niches. These niches offer partial protection from the effects of cytotoxic chemotherapy, otherwise termed cell adhesion-mediated drug resistance. In these experiments, we investigate the apoptotic effects of cytotoxic chemotherapy on HL-60 cell line cultured in a designed 3D AML cell culture model. In this 3D microenvironment, HL-60 cells were co-cultured with ex vivo expanded bone marrow mesenchyaml stem cells in a 3D synthetic scaffold. Aim: To examine the apoptotic effect of cytotoxic chemotherapy on HL-60 co-cultured with human bone marrow mesenchymal stem cells (huBM-MSCs) in 3D conditions. Methods: After several passages, expanded huBM-MSCs were seeded into PGA/PLLA 90/10 copolymer discs, 5-mm in diameter and 2-mm in thickness and allowed to attach to scaffold fibers and to expand over 2 weeks. Then, HL-60 were added and allowed to grow in the 3D culture system for another 10 days. HL-60 cells in 3D culture system were then exposed to doxorubicin given in two concentrations (25 and 50 μM) and incubated for 24 hours. HL-60 were then retrieved applying a combination of mechanical forces and using cell dissociation solution. FITC Annexin V Apoptosis Detection Kit was used to determine apoptosis. Apoptosis was confirmed by TUNEL assay. Proliferation of HL-60 cells in the 3D scaffold was assessed using Ki-67 stain of scaffold's cryosections. All tests were done in triplicates, and untreated HL-60 served as controls for treatment. Comparison was made with HL-60 cells alone and with HL-60 cells growing on a hu-BM-MSC monolayer. SAS version 9.2 (SAS Institute, Inc., 2002–2008) was used for statistical analysis Results: Virtually, all HL-60 cells treated with 25 or 50 μM underwent late apoptosis. Around.03% of HL-60 cells survived 25 μM concentration, none, however, survived 50 μM concentration. In 2D, most of HL-60 cells underwent necrosis, and to lesser extent late apoptosis. In sharp contrast, 17.8% of HL-60 cells survived 25μM concentration, nevertheless, only.27% of HL-60 cells treated with 50 μM concentration survived. The differences in apoptosis patterns between the three groups was statistically significant (P<.0001). Conclusion: compared to traditional cell culture conditions, the designed 3D culture conditions protected a higher percentage of HL-60 cells from undergoing apoptosis and necrosis. Disclosures: No relevant conflicts of interest to declare.