Gillian I. Bell

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.

Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization.

Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes.

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 (ALDHhi) activity, a conserved stem/progenitor cell function. ALDHhi 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 ALDHhi cells demonstrated a proangiogenic transcription profile and were highly enriched for both multipotent myeloid and endothelial colony‐forming cells in vitro. Coculture of ALDHhi 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, ALDHhi cells significantly increased tube‐like cord formation by HUVEC. After induction of acute unilateral hind limb ischemia by femoral artery ligation, transplantation of ALDHhi cells significantly enhanced the recovery of perfusion in ischemic limbs. Despite transient engraftment in the ischemic hind limb, early recruitment of ALDHhi cells into ischemic muscle tissue correlated with increased murine von Willebrand factor blood vessel and CD31+ capillary densities. Thus, UCB ALDHhi 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 ALDHhi mixed progenitor cells. STEM Cells2012;30:2248–2260

Combinatorial Human Progenitor Cell Transplantation Optimizes Islet Regeneration Through Secretion of Paracrine Factors

Transplanted human bone marrow (BM) and umbilical cord blood (UCB) progenitor cells activate islet-regenerative or revascularization programs depending on the progenitor subtypes administered. Using purification of multiple progenitor subtypes based on a conserved stem cell function, high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), we have recently shown that transplantation of BM-derived ALDH(hi) progenitors improved systemic hyperglycemia and augmented insulin secretion by increasing islet-associated proliferation and vascularization, without increasing islet number. Conversely, transplantation of culture-expanded multipotent-stromal cells (MSCs) derived from BM ALDH(hi) cells augmented total beta cell mass via formation of beta cell clusters associated with the ductal epithelium, without sustained islet vascularization. To identify paracrine effectors produced by islet-regenerative MSCs, culture-expanded BM ALDH(hi) MSCs were transplanted into streptozotocin-treated nonobese diabetic/severe combine immune deficient (SCID) mice and segregated into islet-regenerative versus nonregenerative cohorts based on hyperglycemia reduction, and subsequently compared for differential production of mRNA and secreted proteins. Regenerative MSCs showed increased expression of matrix metalloproteases, epidermal growth factor receptor (EGFR)-activating ligands, and downstream effectors of Wnt signaling. Regenerative MSC supernatant also contained increased levels of pro-angiogenic versus pro-inflammatory cytokines, and augmented the expansion of ductal epithelial but not beta cells in vitro. Conversely, co-culture with UCB ALDH(hi) cells induced beta cell but not ductal epithelial cell proliferation. Sequential transplantation of MSCs followed by UCB ALDH(hi) cells improved hyperglycemia and glucose tolerance by increasing beta cell mass associated with the ductal epithelium and by augmenting intra-islet capillary densities. Thus, combinatorial human progenitor cell transplantation stimulated both islet-regenerative and revascularization programs. Understanding the progenitor-specific pathways that modulate islet-regenerative and revascularization processes may provide new approaches for diabetes therapy.

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.

EMILIN‐1 and ILK 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

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

Canagliflozin Improves the Recovery of Blood Flow in an Experimental Model of Severe Limb Ischemia

Canagliflozin, a sodium-glucose cotransporter 2 inhibitor (SGLT2i), was shown to reduce major adverse cardiovascular events in the recent CANVAS (CANagliflozin cardioVascular Assessment Study) program [(1)][1]. Canagliflozin treatment was, however, also associated with a significant increase in the

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 (ALDHhi) stimulate islet regeneration after transplantation into mice with streptozotocin‐induced β cell deletion. However, ALDHhi cells represent a rare progenitor subset and widespread use of UCB ALDHhi cells to stimulate islet regeneration will require progenitor cell expansion without loss of islet regenerative functions. Here we demonstrate that prospectively purified UCB ALDHhi 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 ALDHhi cells diminished as culture time progressed such that total ALDHhi 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 (ALDHlo subset) from less differentiated progeny with high ALDH‐activity (ALDHhi subset). The ALDHhi 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 ALDHlo subset. Notably, bulk cells or ALDHlo cells did not possess the functional capacity to lower hyperglycemia after transplantation into streptozotocin‐treated NOD/SCID mice. However, transplantation of the repurified ALDHhi 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. Stem Cells 2016;34:873–887

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.