Vivo Engraftment Research Articles

Transcriptional Profiling of Skeletal Muscle Stem Cells After In Vivo Engraftment into a Heterologous Niche Environment.

Adult stem cells play a critical role in the maintenance and repair of the organs in which they reside. However, their function is highly dependent on the crosstalk with their niche environment that changes during development and in disease states. The niche provides signals to stem cells to activate, proliferate, self-renew, or remain in quiescence. In skeletal muscle, the niche is perturbed in disease contexts such as aging, muscular dystrophies, and cachexia. Therefore, it is important to develop methods that permit the decoupling of niche-mediated from cell-intrinsic changes that occur in muscle stem cells (MuSCs) in development and disease contexts. With the purpose of determining the effect of the niche environment on the MuSC transcriptome, function, or health, we have coupled an allogeneic stem cell transplantation system, meaning the transplantation of MuSCs from a donor mouse into a recipient host mouse, with Switching Mechanism at 5' End of RNA Template (SMART-Seq) to quantify the effects of the niche on the MuSC transcriptome in vivo. Briefly, MuSCs are isolated from a GFP reporter donor mouse (Pax7-nGFP) and transplanted into the irradiated muscles of immunocompromised allogeneic hosts. The MuSCs are re-isolated by fluorescence-activated cell sorting (FACS) after three weeks of inhabiting the heterologous niche, defined as a niche that is different from their originating niche, and sequencing-ready libraries are created. This method allows for the direct comparison of the transcriptome of stem cells before and after transplantation into a host of a different age, disease status, or genetic background. This method can be used to accurately quantify the direct effect of the niche environment on the stem cell gene expression profile and to decouple cell-intrinsic versus niche-mediated alterations in the stem cell transcriptome. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Allogeneic muscle stem cell transplantation.

M2 Macrophages Drive Resistance to Phagocytosis and Improve Mitochondrial Metabolism in Acute Myeloid Leukemia Facilitating Leukemic Transformation and In Vivo Engraftment

Acute myeloid leukemia (AML) is a heterogeneous disease, which also extends to its tumor microenvironment (TME). Using single cell RNA-seq data (GSE116256), we performed differential gene expression analysis on macrophages from AML patients (AML-derived macrophages, AdMs) compared to healthy bone marrow macrophages. Thus, 306 upregulated genes in AdMs were identified as involved in different biological pathways such as proliferation, inflammation and cellular metabolism. Down-regulated genes (232) were mainly associated with the regulation of apoptosis. Single cell gene set enrichment analysis associated AdMs with an M2-like phenotype and an immunosuppression transcriptional program. We then investigated the macrophage landscape in a large cohort of AML patients by flow cytometry (n = 61) and immunohistochemistry, which revealed a large heterogeneity whereby AML patients with a predominant M2-like TME showed poor prognosis. Next, we evaluated the immune function of AdMs compared to healthy donor-derived macrophages. Thus, we showed that AdMs displayed decreased phagocytic capacity towards AML cells compared to healthy macrophages. Since AdMs might directly descend from the mutated leukemic clones we evaluated whether these mutations could affect the immune function of macrophages. Cord blood-derived CD34+ cells were lentivirally transduced with FLT3-ITD, differentiated towards macrophages, and these FLT3-ITD+ macrophages displayed decreased phagocytic activity. We questioned whether endogenous murine macrophages that are still present in immunodeficient mouse strains that are often used for human studies might form a barrier that needs to be taken to achieve efficient engraftment of human cells. Macrophages were isolated from NSG mice and were used in phagocytosis assays. The percentage of phagocytosed primary AML blasts by murine macrophages ranged between 3.2-51.4%, similar to phagocytosis levels observed when using human macrophages for the same AML samples. While performing these phagocytosis assays, we noted that the cells that remained and were not phagocytosed had an altered appearance and were very viable, as if these cells had gained fitness. We tested this further by exposing leukemic blasts to M2 macrophages for 48 h, after which the remaining cells were analyzed functionally. We observed that the 48 h "training" of primary leukemic blasts significantly enhanced their mitochondrial metabolism (in part mediated via mitochondrial transfer from macrophages to leukemic blasts), enhanced their in vitro and in vivo homing capacity and that they became resistant to phagocytosis. As a most striking example we used primary APL cells that are notoriously difficult to engraft in NSG mice, which we hypothesized could be in line with their high intrinsic susceptibility to macrophage-mediated phagocytosis. APL cells were exposed to M2 macrophages for 48h resulting in two cellular fates: 91.7% of cells were phagocytosed, but the remaining 8.3% APL blasts were able to induce fatal leukemia when transplanted into NSGS mice in all evaluated cases (n = 6), while prior to training these cells did not engraft long-term and were not able to confer disease. We also tested the tumor supportive capacity on M2 macrophages by performing intrafemoral injection in NSGS mice after which non-trained APL blasts were injected intravenously. Again, efficient engraftment and fatal leukemia development were observed, but only in mice co-injected with M2 macrophages. Strategies to target the supportive AdM subpopulation would be of interest. In our scRNA-seq analysis, we identified the metabolic enzyme Nicotinamide phosphoribosyltransferase (NAMPT) to be upregulated in AdMs. Ex vivo studies revealed that while conventional chemotherapeutics, AraC/venetoclax, efficiently target the AML blasts (CD34+/CD117+) with minimum effects on the AdMs, NAMPT inhibition with KPT-9274 was able to efficiently target both the AML blasts and the AdM fractions. Functionally, treatment of M2 macrophages with KPT-9274 shifted their polarization towards an M1 phenotype, with reduced support to AML cells in co-culture assays. In conclusion, we uncover the heterogeneity in the macrophage landscape in AML patients, show the functional relevance of M2-polarized macrophages for leukemic transformation, and provide alternative approaches for targeting aimed at the tumor-supportive microenvironment.

Cold Atmospheric Plasma Suppressed MM In Vivo Engraftment by Increasing ROS and Inhibiting the Notch Signaling Pathway

Multiple myeloma (MM) is the second most common hematologic malignancy. MM stem cells (MMSCs) are thought to be the main causes of in vivo engraftment and eventual recurrence. As a notable new technology, cold atmospheric plasmas (CAPs) show a promising anti-tumor effect, due to their production of various ROS. In this study, we found that different types of plasma could inhibit MM’s ability to form cell colonies, suppress MM in vivo engraftment, and extend survival times. We demonstrated that NAC (a ROS scavenger) could block ROS increases and reverse the inhibition of MM’s cell-colony-formation ability, which was induced by the plasma treatment. By using a stem cell signaling array, we found that the Notch pathway was inhibited by the plasma treatment; this was further confirmed by conducting real-time PCRs of three MM cell lines. Together, these results constitute the first report of plasma treatment inhibiting MM in vivo engraftment and prolonging survival time by suppressing the Notch pathway via ROS regulation.

Engineered M13 Peptide Carrier Promotes Angiogenic Potential of Patient-Derived Human Cardiac Progenitor Cells and In Vivo Engraftment.

Despite promising advances in stem cell-based therapy, the treatment of ischemic cardiovascular diseases remains a big challenge due to both the insufficient in vivo viability of transplanted cells and poor angiogenic potential of stem cells. The goal of this study was to develop therapeutic human cardiac progenitor cells (hCPCs) for ischemic cardiovascular diseases with a novel M13 peptide carrier. In this study, an engineered M13 peptide carrier was successfully generated using a QuikChange Kit. The cellular function of M13 peptide carrier-treated hCPCs was assessed using a tube formation assay and scratch wound healing assay. The in vivo engraftment and cell survival bioactivities of transplanted cells were demonstrated by immunohistochemistry after hCPC transplantation into a myocardial infarction animal model. The engineered M13RGD+SDKP peptide carrier, which expressed RGD peptide on PIII site and SDKP peptide on PVIII site, did not affect morphologic change and proliferation ability in hCPCs. In contrast, hCPCs treated with M13RGD+SDKP showed enhanced angiogenic capacity, including tube formation and migration capacity. Moreover, transplanted hCPCs with M13RGD+SDKP were engrafted into the ischemic region and promoted in vivo cell survival. Our present data provides a promising protocol for CPC-based cell therapy via short-term cell priming of hCPCs with engineered M13RGD+SDKP before cell transplantation for treatment of cardiovascular disease.

Activation of the Receptor Tyrosine Kinase (RET) By GDNF/GFRα1 Improves Cord Blood-Derived HSC in Vitro expansion and In Vivo engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRα1), significantly improves the outgrowth of HSCs invitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (~1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (~1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRα1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRα1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRα1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRα1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRα1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFκB activation and increased CD44 cell surface expression, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRα1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in >10-fold increase in rate of HDR and >120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p<0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p<0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p<0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p<0.001) and >120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p<0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p<0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to >120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Two Long Non-Coding RNAs Are Sufficient to Classify and Significantly Predict In Vivo Engraftment Potential and LSC Properties of Human Acute Myeloid Leukemia Cells

Acute Myeloid Leukemia (AML) is a hematological malignancy with heterogeneous genetics and clinical course. Studies have shown that there is a strong correlation between xenograft in vivo engraftment potential and clinical outcome. Furthermore, such studies have conducted gene expression profiling to identify gene signatures associated with engraftment potential, leukemic stem cell (LSC) property, and prognosis. Most of these analyses were limited to protein coding genes. More recently, advances in next generation sequencing (NGS) have created a paradigm shift on our perception of transcription that, contrary to previous consensus, the human genome is largely transcribed and most of these transcripts do not code for proteins (ncRNA). In the current work, we conducted a comprehensive functional genomics study to identify long ncRNA (lncRNA) that significantly predict in vivo engraftment potential and LSC properties of AML and evaluate their functional and prognostic relevance.We first sorted normal granulocyte-macrophage progenitor (GMP), lymphoid-primed multi-potential progenitor (LMPP) and CD34-subpopulations from leukemic bulk of 15 AML patient samples. Sorted samples were then transplanted into the non-obese diabetic/severe combined immunodeficient Gamma (NSG) mouse model. Only the subpopulations showing robust leukemic engraftment (> 1%) were classified as LSC populations. In parallel, we performed total RNA based RNA-seq using Ovation® Single Cell RNA-Seq System (NuGEN Technologies Inc.) and evaluated both protein coding genes and lncRNAs.In line with previous studies, we observed that the engraftment potential primarily resides within the CD34+ subpopulations (GMPs and LMPPs). LMPPs/GMPs from 8 AML patients engrafted (defined as LSC populations), while subpopulations from the other 7 patients failed to engraft (non-LSC fraction). Using our RNA-seq data, we first conducted a global principal component analysis (PCA) based between group analysis (BGA) based on lncRNA expression levels. Interestingly, we noted that the LMPPs and GMPs primarily cluster based on their engraftment potential rather than cell type. In two subsequent independent analyses, we employed weighted correlation network analysis (WGCNA) and top scoring pairs (tsp) to identify lncRNAs that show significant correlation to engraftment percentages and could also categorically classify samples based on their engraftment status. Results of the WGCNA analysis showed that 830 lncRNAs are statistically significantly correlated to engraftment percentages of subpopulations (p < 0.05). In the tsp analysis, by taking all lncRNAs in the dataset in a pairwise fashion, we identified two lncRNAs that could statistically significantly classify samples into ’LSC’ (engrafters) and ’non-LSCs’ (non-engrafters) (mean classification error ~ 0.19 using bootstrap analysis; statistically significant with p-value < 0.0001 using Monte Carlo Simulation). Of interest here, the two lncRNAs were also in the list of significantly correlated lncRNAs based on the separately conducted WGCNA. Combining the two analyses and using a 3d plot, we looked at the expression levels of two lncRNAs and engraftment percentages of the subpopulations and were able to see that the GMP/LMPP LSCs formed a distinct cluster. The non-LSCs with GMP or LMPP phenotype were distantly clustered from their engrafting counterparts while all the CD34-negative non-LSCs clustered with no overlap to the other groups. Furthermore, a guilt-by-association analysis was carried out to identify potential functional link of the candidate lncRNAs by assessing their correlation to functionally annotated genes. ’JAK-STAT’, ’Hematopoietic lineage’, and ’Toll-like receptor signaling’ were some of the significant pathways (p < 0.05; FDR < 0.25). We also tested some published AML LSC and engraftment associated signatures. The lncRNAs showed negative correlation to the Eppert et al. (2011; Nat. Med.) LSC signature and set of lineage affiliated genes (Goardon et al. 2011; Cancer Cell) including FLT3, NOTCH1, and RUNX1. Taken together, our approach shows that the two lncRNAs we identified sufficiently recapitulate the underlying engraftment potential of AMLs and predict LSC property with significant accuracy. We conclude that these findings highlight the necessity to focus on lncRNAs as key players in clinical and functional studies of AML. DisclosuresBuske:Celltrion, Inc.: Consultancy, Honoraria. Mulaw:NuGEN: Honoraria.

Correction: Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Correction: Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Correction: Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

[This corrects the article on p. e72023 in vol. 8.].

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.

Involvement of Dll1/Notch Interaction In MM Drug Resistance, Clonogenic Growth and In Vivo Engraftment

AbstractAbstract 2966The great challenges in multiple myeloma (MM) treatment are to overcome drug resistance and to prevent relapse. The bone marrow (BM) microenvironment plays a critical role in MM cell growth and survival. The Notch pathway was found to be activated in MM. In this study we aimed at investigating the role of Dll1/Notch interaction in MM clonogenic growth and in vivo engraftment, as well as the role of Notch pathway in BM-induced drug resistance. Dll1 is a Notch ligand expressed in BM stromal cells. Cocultures were performed using murine 5T33MM cells with Dll1 ligand or MS5.Dll1 (Dll1-overexpressing) stromal cells. Notch downstream target genes (Hes1, Hes5, Hey1, Hey2, and HeyL) were investigated for Notch pathway activation, showing that 5T33MM cells expressed Notch target genes and that Hes5 and HeyL were up-regulated both on mRNA (Real-Time PCR) and protein level (western blot) after Dll1/Notch interaction. This up-regulation could be reverted by blocking Notch signaling with DAPT. Moreover, only adhesive cell-cell direct interaction was necessary for MM Notch activation and no soluble factors in the coculture system were involved. Colony-forming-cell (CFC) assays and in vivo engraftment studies were performed to investigate MM clonogenic ability and MM disease initiation. 5T33MM cells cocultured for several days on MS5.Dll1 cells were able to form more tumor colonies compared to 5T33MM cells cocultured on MS5 control cells. Furthermore, these cocultured 5T33MM cells were injected intravenously into naive mice to investigate disease development. Mice injected with Dll1-activated 5T33MM cells developed faster MM disease than those injected with control cells, and also had a higher paraprotein concentration (1.98 ± 0.44 g/dl, n=3) compared to control mice (0.95 ± 0.02 g/dl, n=3). 5T33MM cells pretreated with DAPT for 6 days, to inhibit constitutive Notch pathway activation, had a decreased ability to form colonies in the CFC assay compared to control cells. In addition, mice were injected with 5T33MM cells pretreated with DAPT or DMSO. After 59 days, the DMSO control group became sick with an average M paraprotein of (1.78±0.14g/dl, n=3), while in the DAPT group, all mice were healthy and no M paraprotein could be detected at that time. The DAPT treated group did develop MM disease, but significantly delayed (average of 161 days after injection) with a lower M paraprotein amount (0.35±0.13g/dl, n=3). All these results suggest that the Notch pathway is involved in MM in vitro clonogenic growth and in vivo engraftment. Further studies revealed that Notch pathway activation with Dll1 could induce drug resistance to Bortezomib treatment. Cell viability assay showed that 72.6% of 5T33MM cells stimulated with Dll1 remained alive after Bortezomib treatment compared to only 24.9% alive in IgG control. This protective effect could be reverted by inhibiting Notch pathway with DAPT. After cocultures of MM cells with MS5.Dll1 stromal cells, we detected an increased percentage of CD138- MM cells and a down-regulation of CD138 mRNA expression compared to cocultures with MS5 stromal cells. This could be reverted by preventing cell-cell interaction using transwells or by adding DAPT. Similar results were found with human RPMI-8226 cells. Matsui et al. (Cancer Res 2008; 68: (1) 190–197) and our lab demonstrated that CD138- MM cells were less sensitive to drugs than CD138+ MM cells. Interestingly, CD138- MM cells showed a higher level of Notch activation than CD138+ MM cells, suggesting that Notch activation is involved in the protection of CD138- MM cells from drug induced cell death.In conclusion, our data show that Dll1/Notch pathway activation could promote MM clonogenic growth and accelerate MM development. Inhibiting Notch pathway can significantly suppress MM initiation and delay the engraftment. Moreover, Dll1/Notch interaction could also induce drug resistance to Bortezomib, by altering MM cells to a more resistant CD138- phenotype. Disclosures:No relevant conflicts of interest to declare.

Modulation of Mesenchymal Stem Cell MHC-I Complex Increases Engraftment In Vivo.

AbstractAbstract 1457Mesenchymal stem cells (MSC) are good candidates for cell therapies due to their immunomodulatory properties, ability to home to/engraft damaged tissues, and potential to differentiate into different cell types. However, when transplanted (Tx) in an allogeneic setting, MSC can elicit an immune response, activating the recipient's cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells, resulting in rejection of the Tx cells and reduced therapeutic efficacy. Human cytomegalovirus (HCMV, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins that downregulate MHC-I surface expression. These proteins are coded for by the unique short regions (US) 2, 3, 6 and 11 of HCMV's genome. We have previously shown that when MSC are transduced with retroviral vectors encoding each one of these US proteins, US6 and US11 were the most effective in reducing MSC's HLA-I surface expression and allogeneic CTL recognition and proliferation. However, HLA-I downregulation may render MSC transduced with US6 (MSC-US6) and US11 (MSC-US11) more susceptible to NK killing, undermining MSC's inherent ability to inhibit function of allogeneic NK cells. Here, we first investigated the role of US6 or US11 on MSC allorecognition by NK cells, and on MSC in vivo engraftment capability. NK killing assays demonstrated that US11 generated the most protective effect at the highest NK concentration (E:T ratio 20:1) (% specific lysis for MSC-US6: 60.4 ± 5.7 %; MSC-US11: 45.5 ± 2.4 % vs. MSC: 88.5 ± 3.4 % respectively). However, at an E:T ratio of 10:1 and 5:1 US11 produced the same degree of protection as US6 (E:T ratio of 10:1; % specific lysis for MSC-US6: 30.1 ± 5.6 %; MSC-US11: 26.3 ± 1.9 % vs. MSC: 54.7 ± 1.9 %); (E:T ratio 5:1; % specific lysis for MSC-US6: 11.9 ± 4.2; MSC-US11: 13.4 ± 2.3; vs. MSC: 25.5 ± 4 respectively). Only at an E:T ratio of 1:1 were US6 and US11 similar to untransduced MSCs (% specific lysis for MSC-US6: 4.7 ± 1.6; MSC-US11: 2.1 ± 0.5; vs. MSC: 4.9 ± 1.8; respectively) in terms of inhibition of NK killing. We also studied the role of US6 and 11 on the expression of beta-2-microglobulin (b2m) and other HLA-I molecules, and we found that US6 reduced b2m by 87± 2 % and HLA-G1 by 44±4.7 %, while US11 reduced b2m by 70± 0.6 % but increased HLA-G1 expression by 176.6±1.9 %. Therefore, the increase in HLA-G1 expression induced by US11 may explain the decrease in NK killing observed in the MSC-US11 cells. Furthermore, we investigated whether US6 or US11 could play a role in mediating complement resistance. While US6 increased the expression of CD59 in transduced cells (Mean fluorescence intensity (MFI) increased by 123.3±1), US11 increased the number of cells expressing CD59 by 121.4 ± 0.8 %, but did not modify their MFI. We next compared the in vivo engraftment potential of MSC, MSC-US6 and MSC-US11 by Tx 5.6×10^4 of each cell population into fetal sheep at 60 days of gestation (n=6). Since we have previously reported the ability of MSC to generate liver cells, we first investigated whether the expression of US6 and 11 would allow higher levels of liver engraftment and hepatocyte formation when compared to MSC (MSC-E) transduced with a retroviral vector encoding only NPT-II. Two months after Tx, liver tissues were collected and stained with NPT-II antibody. This revealed that US6 and US11 increased engraftment efficiency by 241% for MSC-US6 and 277% for MSC-US11 (MSC-E: 5.3 ± 0.4 %, MSC-US6: 12.8 ± 0.9 % and MSC-US:11 14.7 ± 0.8 %). Despite the higher level of liver engraftment seen with MSC-US6 and MSC-US11, co-expression of NPT-II and albumin (MSC-US6: 57% MSC-US1: 50% MSC-E: 75%) or NPT-II and Ov-6 was found at significantly lower levels in MSC-US11 and MSC-US6 Tx animals than in those Tx with MSC-E. Nevertheless, similar numbers of NPT-II/CD34 double-positive cells were found in the liver of MSC-US6 and MSC-US11 Tx animals when compared to MSC-E alone. In conclusion, engineering MSC to over-express US6 or US11 is an effective way to reduce CTL proliferation, NK killing and destruction of engrafted cells by the complement membrane attack complex. In agreement with the in vitro studies, transplantation of these cells into a large animal sheep model resulted in significantly higher levels of overall cell engraftment, but not differentiation towards a hepatocytic phenotype. Studies are underway to determine the mechanism by which HCMV proteins are interfering with MSC differentiation. Disclosures:No relevant conflicts of interest to declare.

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

BackgroundCord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34+ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.Methodology/Principal FindingsCB CD34+ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34+ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.Conclusion/SignificanceOur data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34+ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.

485. Long-Term In Vivo Engraftment and Transgene Expression from CD34+ Human Hematopoietic Stem Cells Transduced with Pseudotyped rAAV

The search for the ideal vector for stem cell gene therapy continues as recognized problems remain with all existing viral vectors. The recent identification of over 100 new serotypes of AAV significantly expands the repertoire of available gene transfer vectors. Additionally, the capacity to pseudotype rAAV2 genomes in novel capsids facilitates evaluation. We and others have analyzed rAAV2-mediated gene transfer into hematopoietic stem cells (HSC). However, despite efficient gene-marking of primitive HSCs, there are identified limitations to transgene expression especially at early time points.

Establishment of a Waldenstrom's Macroglobulinemia Cell Line (BCWM.1) with Productive In Vivo Engraftment in SCID-hu Mice.

Waldenstrom's Macroglobulinemia (WM) is incurable B-cell malignancy characterized by the infiltration of the bone marrow infiltration with lymphoplasmacytic cells (LPC) and the presence of an IgM monoclonal gammopathy. A significant impairment in understanding the biology and advancing therapeutics for WM has been the lack of a representative cell line and animal model. We therefore report on work accomplished in our laboratories in establishing the BWMC.1 cell line, which was derived from long term culture of CD19+ selected lymphoplasmacytic cells isolated from the bone marrow (BM) aspirate of a previously untreated 55-year-old female patient with IgMk secreting WM. BCWM.1 cells morphologically resembled LPC and had prominent nucleoli and ample cytoplasm by Wright-Giemsa staining, and were propagated in RPMI 1640 medium supplemented with 10% FBS. Phenotypic characterization by flow cytometric analysis demonstrated typical WM LPC characteristics: CD5−, CD10−, CD19+, CD20+, CD23+, CD27−, CD38+, CD138+, CD40+, CD52+, CD70+, CD117+, cIgM+, cIgG−, cIgA−, ck+, cl−. BCWM.1 cells also expressed the survival proteins APRIL and B-Lymphocyte Stimulator (B-LYS), and their receptors TACI, BCMA and BAFF-R. ELISA studies demonstrated secretion of IgMk in culture. Karyotypic and M-FISH studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements through FR1c/JHc amplification of genomic DNA. BCWM.1 cells were positive for LMP1 expression by histochemical staining and PCR analysis. Importantly, inoculation of BCWM.1 cells directly into the human BM milieu of SCID-hu mice resulted in engraftment of tumor cells and serum detection of human IgMk in four of five SCID-hu mice within two weeks. In contrast, subcutaneous injection of BCWM.1 cells in SCID mice did not lead to engraftment, suggesting a role for the human BM microenvironment in supporting the expansion of BCWM.1 cells. These studies therefore support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the SCID-hu mouse model may be used as a convenient model for studies focused on both the pathogenesis and development of targeted therapies for WM.

SPRY1 Is a Negative Regulator of Long-Term In Vivo Engraftment and Ex Vivo Expansion of Primitive Human Umbilical Cord Blood Cells.

Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p<0.05) and CFCs (SPRY1-LV = 5.7 ± 1.2 fold; GFP-LV = 20.8 ± 14.9 fold; n = 3), although it had no effect on expansion of CD34+CD38− cells. We are currently investigating potential mechanisms for the observed affects of SPRY1 on primitive hematopoietic cells, paying particular attention to the possible effects of SPRY1 expression on “early-acting” hematopoietic cytokines and growth factors that activate RTKs - including FGF1, FGF2, VEGF, SCF, FLT3L, and ANGPT1. In conclusion, enforced expression of SPRY1 negatively regulates primitive hematopoietic cell engraftment in vivo and expansion in vitro, thereby presenting the first example of a role for a Sprouty family member, SPRY1, in primitive human hematopoietic cell function. Moreover, this data further validates the use of model organisms, such as zebrafish, for evaluating the functional roles of transcripts identified in large-scale gene expression profiling experiments in mice and humans.