Targeting the MR1-MAIT cell axis improves vaccine efficacy and affords protection against viral pathogens.

Mucosa-associated invariant T (MAIT) cells are MR1-restricted, innate-like T lymphocytes with tremendous antibacterial and immunomodulatory functions. Additionally, MAIT cells sense and respond to viral infections in an MR1-independent fashion. However, whether they can be directly targeted in immunization strategies against viral pathogens is unclear. We addressed this question in multiple wild-type and genetically altered but clinically relevant mouse strains using several vaccine platforms against influenza viruses, poxviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), a riboflavin-based MR1 ligand of bacterial origin, can synergize with viral vaccines to expand MAIT cells in multiple tissues, reprogram them towards a pro-inflammatory MAIT1 phenotype, license them to bolster virus-specific CD8+ T cell responses, and potentiate heterosubtypic anti-influenza protection. Repeated 5-OP-RU administration did not render MAIT cells anergic, thus allowing for its inclusion in prime-boost immunization protocols. Mechanistically, tissue MAIT cell accumulation was due to their robust proliferation, as opposed to altered migratory behavior, and required viral vaccine replication competency and Toll-like receptor 3 and type I interferon receptor signaling. The observed phenomenon was reproducible in female and male mice, and in both young and old animals. It could also be recapitulated in a human cell culture system in which peripheral blood mononuclear cells were exposed to replicating virions and 5-OP-RU. In conclusion, although viruses and virus-based vaccines are devoid of the riboflavin biosynthesis machinery that supplies MR1 ligands, targeting MR1 enhances the efficacy of vaccine-elicited antiviral immunity. We propose 5-OP-RU as a non-classic but potent and versatile vaccine adjuvant against respiratory viruses.

Characterization of a Vimentinhigh /Nestinhigh proteome and tissue regenerative secretome generated by human pancreas-derived mesenchymal stromal cells.

Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.

Characterization of extracellular vesicles derived from mesenchymal stromal cells by surface-enhanced Raman spectroscopy.

Extracellular vesicles (EVs) are secreted by all cells into bodily fluids and play an important role in intercellular communication through the transfer of proteins and RNA. There is evidence that EVs specifically released from mesenchymal stromal cells (MSCs) are potent cell-free regenerative agents. However, for MSC EVs to be used in therapeutic practices, there must be a standardized and reproducible method for their characterization. The detection and characterization of EVs are a challenge due to their nanoscale size as well as their molecular heterogeneity. To address this challenge, we have fabricated gold nanohole arrays of varying sizes and shapes by electron beam lithography. These platforms have the dual purpose of trapping single EVs and enhancing their vibrational signature in surface-enhanced Raman spectroscopy (SERS). In this paper, we report SERS spectra for MSC EVs derived from pancreatic tissue (Panc-MSC) and bone marrow (BM-MSC). Using principal component analysis (PCA), we determined that the main compositional differences between these two groups are found at 1236, 761, and 1528 cm-1, corresponding to amide III, tryptophan, and an in-plane -C=C- vibration, respectively. We additionally explored several machine learning approaches to distinguish between BM- and Panc-MSC EVs and achieved 89 % accuracy, 89 % sensitivity, and 88 % specificity using logistic regression.

Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration.

Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into ß-like cells after near complete ß-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous ß-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of ß-cell phenotype by α-cells, and accelerated ß-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of ß-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.

Inhibition of Aldehyde Dehydrogenase-Activity Expands Multipotent Myeloid Progenitor Cells with Vascular Regenerative Function.

Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDHhi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDHhi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDHhi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDHhi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDHhi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDHhi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736.

Proteomic characterisation reveals active Wnt-signalling by human multipotent stromal cells as a key regulator of beta cell survival and proliferation.

AIMS/HYPOTHESIS: Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. METHODS: Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSCR) vs nonregenerative (MSCNR) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSCNR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. RESULTS: MSCR showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSCNR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSCR maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSCNR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSCNR samples increased the accumulation of nuclear ß-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. CONCLUSIONS/INTERPRETATION: Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.

Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.

Human umbilical cord blood (UCB) hematopoietic progenitor cells (HPC) purified for high aldehyde dehydrogenase activity (ALDH(hi) ) stimulate islet regeneration after transplantation into mice with streptozotocin-induced ß cell deletion. However, ALDH(hi) cells represent a rare progenitor subset and widespread use of UCB ALDH(hi) cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDH(hi) cells expand efficiently under serum-free, xeno-free conditions with minimal growth factor supplementation. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, kinetic analyses over 9 days revealed the frequency of ALDH(hi) cells diminished as culture time progressed such that total ALDH(hi) cell number was maximal (increased 3-fold) at day 6. Subsequently, day 6 expanded cells (bulk cells) were sorted after culture to reselect differentiated progeny with low ALDH-activity (ALDH(lo) subset) from less differentiated progeny with high ALDH-activity (ALDH(hi) subset). The ALDH(hi) subset retained primitive cell surface marker coexpression (32.0% ± 7.0% CD34(+) /CD38(-) cells, 37.0% ± 6.9% CD34(+) /CD133(+) cells), and demonstrated increased hematopoietic colony forming cell function compared with the ALDH(lo) subset. Notably, bulk cells or ALDH(lo) cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin-treated NOD/SCID mice. However, transplantation of the repurified ALDH(hi) subset significantly reduced hyperglycemia, improved glucose tolerance, and increased islet-associated cell proliferation and capillary formation. Thus, expansion and delivery of reselected UCB cells that retain high ALDH-activity after short-term culture represents an improved strategy for the development of cellular therapies to enhance islet regeneration in situ.

Brief Report: Elastin Microfibril Interface 1 and Integrin-Linked Protein Kinase Are Novel Markers of Islet Regenerative Function in Human Multipotent Mesenchymal Stromal Cells.

Multipotent mesenchymal stromal cell (MSC) transplantation is proposed as a novel therapy for treating diabetes by promoting the regeneration of damaged islets. The clinical promise of such treatments may be hampered by a high degree of donor-related variability in MSC function and a lack of standards for comparing potency. Here, we set out to identify markers of cultured human MSCs directly associated with islet regenerative function. Stromal cultures from nine separate bone marrow donors were demonstrated to have differing capacities to reduce hyperglycemia in the NOD/SCID streptozotocin-induced diabetic model. Regenerative (R) and non-regenerative (NR) MSC cultures were directly compared using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics. A total of 1,410 proteins were quantified resulting in the identification of 612 upregulated proteins and 275 downregulated proteins by ± 1.2-fold in R-MSC cultures. Elastin microfibril interface 1 (EMILIN-1), integrin-linked protein kinase (ILK), and hepatoma-derived growth factor (HDGF) were differentially expressed in R-MSCs, and Ingenuity Pathway Analyses revealed each candidate as known regulators of integrin signaling. Western blot validation of EMILIN-1, ILK, and HDGF not only showed significantly higher abundance levels in R-MSCs, as compared with NR-MSCs, but also correlated with passage-induced loss of islet-regenerative potential. Generalized estimating equation modeling was applied to examine the association between each marker and blood glucose reduction. Both EMILIN-1 and ILK were significantly associated with blood glucose lowering function in vivo. Our study is the first to identify EMILIN-1 and ILK as prospective markers of islet regenerative function in human MSCs. Stem Cells 2016;34:2249-2255.

PU.1 opposes IL-7-dependent proliferation of developing B cells with involvement of the direct target gene bruton tyrosine kinase.

Deletion of genes encoding the E26 transformation-specific transcription factors PU.1 and Spi-B in B cells (CD19-CreΔPB mice) leads to impaired B cell development, followed by B cell acute lymphoblastic leukemia at 100% incidence and with a median survival of 21 wk. However, little is known about the target genes that explain leukemogenesis in these mice. In this study we found that immature B cells were altered in frequency in the bone marrow of preleukemic CD19-CreΔPB mice. Enriched pro-B cells from CD19-CreΔPB mice induced disease upon transplantation, suggesting that these were leukemia-initiating cells. Bone marrow cells from preleukemic CD19-CreΔPB mice had increased responsiveness to IL-7 and could proliferate indefinitely in response to this cytokine. Bruton tyrosine kinase (BTK), a negative regulator of IL-7 signaling, was reduced in preleukemic and leukemic CD19-CreΔPB cells compared with controls. Induction of PU.1 expression in cultured CD19-CreΔPB pro-B cell lines induced Btk expression, followed by reduced STAT5 phosphorylation and early apoptosis. PU.1 and Spi-B regulated Btk directly as shown by chromatin immunoprecipitation analysis. Ectopic expression of BTK was sufficient to induce apoptosis in cultured pro-B cells. In summary, these results suggest that PU.1 and Spi-B activate Btk to oppose IL-7 responsiveness in developing B cells.

Umbilical cord blood-derived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury.

Umbilical cord blood (UCB) represents a readily available source of hematopoietic and endothelial precursors at early ontogeny. Understanding the proangiogenic functions of these somatic progenitor subtypes after transplantation is integral to the development of improved cell-based therapies to treat ischemic diseases. We used fluorescence-activated cell sorting to purify a rare (<0.5%) population of UCB cells with high aldehyde dehydrogenase (ALDH(hi) ) activity, a conserved stem/progenitor cell function. ALDH(hi) cells were depleted of mature monocytes and T- and B-lymphocytes and were enriched for early myeloid (CD33) and stem cell-associated (CD34, CD133, and CD117) phenotypes. Although these cells were primarily hematopoietic in origin, UCB ALDH(hi) cells demonstrated a proangiogenic transcription profile and were highly enriched for both multipotent myeloid and endothelial colony-forming cells in vitro. Coculture of ALDH(hi) cells in hanging transwells promoted the survival of human umbilical vein endothelial cells (HUVEC) under growth factor-free and serum-free conditions. On growth factor depleted matrigel, ALDH(hi) cells significantly increased tube-like cord formation by HUVEC. After induction of acute unilateral hind limb ischemia by femoral artery ligation, transplantation of ALDH(hi) cells significantly enhanced the recovery of perfusion in ischemic limbs. Despite transient engraftment in the ischemic hind limb, early recruitment of ALDH(hi) cells into ischemic muscle tissue correlated with increased murine von Willebrand factor blood vessel and CD31+ capillary densities. Thus, UCB ALDH(hi) cells represent a readily available population of proangiogenic progenitors that promote vascular regeneration. This work provides preclinical justification for the development of therapeutic strategies to treat ischemic diseases using UCB-derived ALDH(hi) mixed progenitor cells.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity.

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model.

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and ß cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Peptide-modified methacrylated glycol chitosan hydrogels as a cell-viability supporting pro-angiogenic cell delivery platform for human adipose-derived stem/stromal cells.

Cell-based therapies involving the injection of adipose-derived stem/stromal cells (ASCs) within rationally designed biomaterials are a promising approach for stimulating angiogenesis. With this focus, the current work explored the effects of incorporating integrin-binding RGD or IKVAV peptides within in situ-gelling N-methacrylate glycol chitosan (MGC) hydrogels on the response of encapsulated human ASCs. Initial studies focused on hydrogel characterization to validate that the MGC, MGC-RGD, and MGC-IKVAV hydrogels had similar biomechanical properties. ASC viability following encapsulation and culture under 2% O2 was significantly impaired in the MGC-IKVAV group relative to the MGC and MGC-RGD groups. In contrast, sustained viability, along with enhanced cell spreading and metabolic activity were observed in the MGC-RGD group. Investigation of angiogenic transcription suggested that the incorporation of the peptide groups did not substantially alter the pro-angiogenic gene expression profile of the encapsulated ASCs after 7 days of culture under 2% O2. Consistent with the in vitro findings, preliminary in vivo characterization following subcutaneous implantation into NOD/SCID mice showed that ASC retention was enhanced in the MGC-RGD hydrogels relative to the MGC-IKVAV group at 14 days. Further, the encapsulated ASCs in the MGC and MGC-RGD groups promoted murine CD31+ endothelial cell recruitment to the peri-implant region. Overall, the results indicate that the MGC-RGD and MGC hydrogels are promising platforms for ASC delivery, and suggest that strategies that support long-term ASC viability can augment in vivo angiogenesis through paracrine mechanisms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 571-585, 2019.

Inhibition of Retinoic Acid Production Expands a Megakaryocyte-Enriched Subpopulation with Islet Regenerative Function.

Islet regeneration is stimulated after transplantation of human umbilical cord blood (UCB) hematopoietic progenitor cells with high aldehyde dehydrogenase (ALDH)-activity into NOD/SCID mice with streptozotocin (STZ)-induced ß cell ablation. ALDHhi progenitor cells represent a rare subset within UCB that will require expansion without the loss of islet regenerative functions for use in cell therapies. ALDHhi cells efficiently expand (>70-fold) under serum-free conditions; however, high ALDH-activity is rapidly diminished during culture coinciding with emergence of a committed megakaryocyte phenotype CD41+/CD42+/CD38+. ALDH-activity is also the rate-limiting step in retinoic acid (RA) production, a potent driver of hematopoietic differentiation. We have previously shown that inhibition of RA production during 9-day cultures, using diethylaminobenzaldehyde (DEAB) treatment, enhanced the expansion of ALDHhi cells (>20-fold) with vascular regenerative paracrine functions. Herein, we sought to determine if DEAB-treatment also expanded ALDHhi cells that retain islet regenerative function following intrapancreatic transplantation into hyperglycemic mice. After DEAB-treatment, expanded ALDHhi cell subset was enriched for CD34+/CD38- expression and demonstrated enhanced myeloid multipotency in vitro compared to the ALDHlo cell subset. Unfortunately, DEAB-treated ALDHhi cells did not support islet regeneration after transplantation. Conversely, expanded ALDHlo cells from DEAB-treated conditions reduced hyperglycemia, and increased islet number and cell proliferation in STZ-induced hyperglycemic NOD/SCID mice. DEAB-treated ALDHlo cells were largely committed to a CD41+/CD42+ megakaryocyte phenotype. Collectively, this study provides preliminary evidence that committed cells of the megakaryocyte-lineage support endogenous islet regeneration and/or function, and the retention of high ALDH-activity did not coincide with islet regenerative function after expansion under serum-free culture conditions.

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDHhi), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDHhi cells available for transplantation compared with untreated controls. Newly expanded ALDHhi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDHhi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of ß-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating ß-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDHhi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDHhi cells without the loss of islet regenerative functions.

Quantitative Proteomics Evaluation of Human Multipotent Stromal Cell for ß Cell Regeneration.

Human multipotent stromal cells (hMSCs) are one of the most versatile cell types used in regenerative medicine due to their ability to respond to injury. In the context of diabetes, it has been previously shown that the regenerative capacity of hMSCs is donor specific after transplantation into streptozotocin (STZ)-treated immunodeficient mice. However, in vivo transplantation models to determine regenerative potency of hMSCs are lengthy, costly, and low throughput. Therefore, a high-throughput quantitative proteomics assay was developed to screen ß cell regenerative potency of donor-derived hMSC lines. Using proteomics, we identified 16 proteins within hMSC conditioned media that effectively identify ß cell regenerative hMSCs. This protein signature was validated using human islet culture assay, ELISA, and the potency was confirmed by recovery of hyperglycemia in STZ-treated mice. Herein, we demonstrated that quantitative proteomics can determine sample-specific protein signatures that can be used to classify previously uncharacterized hMSC lines for ß cell regenerative clinical applications.

Driver mutations in Janus kinases in a mouse model of B-cell leukemia induced by deletion of PU.1 and Spi-B.

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with recurrent mutations that occur in cancer-initiating cells. There is a need to understand how driver mutations influence clonal evolution of leukemia. The E26-transformation-specific (ETS) transcription factors PU.1 and Spi-B (encoded by Spi1 and Spib) execute a critical role in B-cell development and serve as complementary tumor suppressors. Here, we used a mouse model to conditionally delete Spi1 and Spib genes in developing B cells. These mice developed B-ALL with a median time to euthanasia of 18 weeks. We performed RNA and whole-exome sequencing (WES) on leukemias isolated from Mb1-CreΔPB mice and identified single nucleotide variants (SNVs) in Jak1, Jak3, and Ikzf3 genes, resulting in amino acid sequence changes. Jak3 mutations resulted in amino acid substitutions located in the pseudo-kinase (R653H, V670A) and in the kinase (T844M) domains. Introduction of Jak3 T844M into Spi1/Spib-deficient precursor B cells was sufficient to promote proliferation in response to low IL-7 concentrations in culture, and to promote proliferation and leukemia-like disease in transplanted mice. We conclude that mutations in Janus kinases represent secondary drivers of leukemogenesis that cooperate with Spi1/Spib deletion. This mouse model represents a useful tool to study clonal evolution in B-ALL.

Expansion of Umbilical Cord Blood Aldehyde Dehydrogenase Expressing Cells Generates Myeloid Progenitor Cells that Stimulate Limb Revascularization.

Uncompromised by chronic disease-related comorbidities, human umbilical cord blood (UCB) progenitor cells with high aldehyde dehydrogenase activity (ALDHhi cells) stimulate blood vessel regeneration after intra-muscular transplantation. However, implementation of cellular therapies using UCB ALDHhi cells for critical limb ischemia, the most severe form of severe peripheral artery disease, is limited by the rarity (<0.5%) of these cells. Our goal was to generate a clinically-translatable, allogeneic cell population for vessel regenerative therapies, via ex vivo expansion of UCB ALDHhi cells without loss of pro-angiogenic potency. Purified UCB ALDHhi cells were expanded >18-fold over 6-days under serum-free conditions. Consistent with the concept that ALDH-activity is decreased as progenitor cells differentiate, only 15.1% ± 1.3% of progeny maintained high ALDH-activity after culture. However, compared to fresh UCB cells, expansion increased the total number of ALDHhi cells (2.7-fold), CD34+ /CD133+ cells (2.8-fold), and hematopoietic colony forming cells (7.7-fold). Remarkably, injection of expanded progeny accelerated recovery of perfusion and improved limb usage in immunodeficient mice with femoral artery ligation-induced limb ischemia. At 7 or 28 days post-transplantation, mice transplanted with expanded ALDHhi cells showed augmented endothelial cell proliferation and increased capillary density compared to controls. Expanded cells maintained pro-angiogenic mRNA expression and secreted angiogenesis-associated growth factors, chemokines, and matrix modifying proteins. Coculture with expanded cells augmented human microvascular endothelial cell survival and tubule formation under serum-starved, growth factor-reduced conditions. Expanded UCB-derived ALDHhi cells represent an alternative to autologous bone marrow as an accessible source of pro-angiogenic hematopoietic progenitor cells for the refinement of vascular regeneration-inductive therapies. Stem Cells Translational Medicine 2017;6:1607-1619.

Transplantation models to characterize the mechanisms of stem cell-induced islet regeneration.

This unit describes our current knowledge regarding the isolation human bone marrow-derived progenitor cells for the paracrine stimulation of islet regeneration after transplantation into immunodeficient mouse models of diabetes. By using high aldehyde dehydrogenase (ALDH(hi) ) activity, a conserved function in multiple stem cell lineages, a mixed population of hematopoietic, endothelial, and mesenchymal progenitor cells can be efficiently purified using flow cytometry. We describe in vitro approaches to characterize and expand these distinct cell types. Importantly, these cell types can be transplanted into immunodeficient mice rendered beta-cell deficient by streptozotocin (STZ) treatment, in order monitor functional recovery from hyperglycemia and to characterize endogenous islet regeneration via paracrine mechanisms. Herein, we provide detailed protocols for: (1) isolation and characterization of ALDH(hi) cells for the establishment of hematopoietic and multipotent-stromal progenitor lineages; (2) intravenous and intrapancreatic transplantation of human stem cell subtypes for the quantification of glycemic recovery in STZ-treated immunodeficient mice; and (3) immunohistochemical characterization of islet recovery via the stimulation of islet neogenic, beta-cell proliferative, and islet revascularization programs. Collectively, these systems can be used to support the pre-clinical development of human progenitor cell-based therapies to treat diabetes via islet regeneration.