Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration
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We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDHhi), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDHhi progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli.
Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDHlo) vs ALDHhi. UCB ALDHlo or ALDHhi cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDHlo or ALDHhi cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ.
UCB-derived ALDHhi cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDHhi cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDHhi cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDHhi cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass.
iPan delivery of UCB ALDHhi cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.
KeywordsAldehyde dehydrogenase Beta cell proliferation Cell therapy Diabetes Intrapancreatic transplantation Islet regeneration Islet revascularisation Umbilical cord blood
Non-obese diabetic/severe combined immune-deficient
Umbilical cord blood
von Willebrand factor
Residual islet function in diabetes patients with disease duration of >50 years suggests that the stimulation of islet regeneration may represent a viable strategy for diabetes treatment . There exists increasing evidence that bone marrow (BM)-derived cells promote islet recovery after transplantation [2, 3, 4], and we have recently shown that the mechanisms of islet regeneration are modulated by the progenitor subtypes administered . While transplanted human multipotent stromal cells stimulated the formation of small islets associated with ducts, i.v. transplanted BM with high aldehyde dehydrogenase (ALDH) activity (ALDHhi) induced islet cell proliferation and led to the recovery of larger and highly perfused islets . As a readily available alternative to BM, we postulated that transplantation of umbilical cord blood (UCB)-derived ALDHhi cells would stimulate islet expansion and vascularisation. Since human cell recruitment to the pancreas is inefficient following i.v. transplantation , we intended to determine if UCB ALDHhi cells delivered directly to the pancreas would permit timely exposure to regenerative stimuli and potentiate the recovery of islet function.
Cell isolation and characterisation
Human UCB was obtained by venipuncture after informed consent at the London Health Sciences Centre. Within 24 h, mononuclear cells were isolated by Ficoll-Hypaque centrifugation, and cells with low ALDH activity (ALDHlo), as well as ALDHhi cells, were purified by cell sorting based on ALDH activity using Aldefluor reagent (Stem Cell Technologies, Vancouver, BC, Canada) as previously described . ALDHlo and ALDHhi cells were characterised for mature haematopoietic and primitive progenitor marker expression, and colony-forming cell (CFC) assays were performed for haematopoietic, endothelial and multipotent stromal progenitor functions as previously described .
Transplantation of hyperglycaemic mice
Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice (Jackson Laboratory, Bar Harbor, ME, USA) were injected with streptozotocin (STZ) 35 mg/kg per day i.p. on days 1–5. At day 10, mice were sublethally irradiated (300 cGy) to reduce residual innate immunity and transplanted by tail vein or intrapancreatic (iPan) injection with PBS or 2 × 105 ALDHlo or ALDHhi cells from a fresh UCB sample. For iPan injections, mice were anaesthetised, the pancreas and spleen exposed, and cells were microinjected (10 μl) into the splenic portion of the pancreas. Non-fasted blood glucose was monitored weekly. Twenty-four hours prior to being killed, each mouse received 200 μg 5-ethynyl-2′-deoxyuridine (EdU), and a fasted (4 h) glucose tolerance test (2.0 g/kg glucose) was performed for a duration of 2 h. Serum was collected for insulin ELISA (Alpco, Salem, NH, USA). BM and pancreas were analysed for human cells by flow cytometry as previously described [5, 7].
Frozen pancreas sections were stained for immunofluorescent analyses to detect murine insulin with human cell engraftment (HLA-A,B,C), blood vessel density (von Willebrand factor; vWF) and EdU incorporation as previously described .
UCB ALDHhi cells possessed haematopoietic and endothelial progenitor phenotypes and functions
We first characterised UCB ALDHlo vs ALDHhi cells for cell surface marker expression and for haematopoietic, endothelial and multipotent stromal colony formation in vitro. Compared with UCB ALDHlo cells that primarily expressed lymphocyte markers, ALDHhi cells highly expressed myeloid (CD33) and haematopoietic/endothelial progenitor markers (CD34, CD117, CD133; see electronic supplementary material [ESM] Table 1). In contrast to BM ALDHhi cells that possessed CFC capacity for all three-progenitor lineages , UCB ALDHhi cells were enriched for multipotent haematopoietic and endothelial CFC (ESM Fig. 1a–e), but did not establish multipotent stromal colonies (ESM Fig. 1c, f). Thus, UCB ALDHhi cells represent a mix of early myeloid cells and haematopoietic progenitors with endothelial progenitor content.
iPan delivery of UCB ALDHhi cells improved endocrine function
iPan delivery of UCB ALDHhi cells increased islet number, size and vascularisation
We postulated that iPan delivery of UCB ALDHhi cells would augment pancreas islet content. Compared with delivery-matched PBS or ALDHlo cells, iPan injection of ALDHhi cells increased both the number and size of islets, and augmented total beta cell mass (Fig. 1g–i). Furthermore, islet size and total beta cell mass were increased compared with i.v. injection of ALDHhi cells (*p < 0.05). Although i.v. delivery of ALDHhi cells modestly increased islet-associated vascularisation (Fig. 1j, l), iPan delivery of ALDHhi cells further improved islet vascularisation (Fig. 1k, l). These effects were observed despite low-frequency human cell engraftment in the pancreas of mice i.v. injected (four of nine mice) or iPan injected (four of five mice) with ALDHhi cells at day 42 (ESM Fig. 2a–b). By contrast, ALDHlo cells were rarely detected in the pancreas after i.v. or iPan injection. Only ALDHhi cell i.v. transplanted mice showed haematopoietic reconstitution in the murine BM (ESM Fig. 2c–d). Collectively, these data suggested that further experiments were warranted to characterise islet regenerative processes in relation to human cell pancreatic engraftment at early time points (days 14–17).
iPan-injected UCB ALDHhi cells surrounded islets and stimulated islet cell proliferation
Next we assessed islet-associated cell proliferation using EdU labelling 24 h prior to sacrifice. While islet-associated proliferation was minimal in mice iPan injected with ALDHlo cells (ESM Fig. 2f), mice iPan injected with ALDHhi cells demonstrated EdU labeling in both insulin− (arrowheads) and insulin+ cells (arrows, Fig. 2d, e). By day 17, the percentage of EdU+ cells within islets was increased (Fig. 2f), as was the frequency of insulin+ EdU+ cells (ALDHhi 29.5 ± 3.4% vs ALDHlo 6.3 ± 1.2%; *p < 0.05). Corresponding to islet proliferation at early time points, iPan ALDHhi transplanted mice showed an increase in non-fasted serum insulin (Fig. 2g, i). However, serum insulin levels remained below threefold lower than citric acid buffer controls. Although iPan transplanted mice did not demonstrate a measurable response to glucose bolus at day 14 (Fig. 2h), iPan ALDHhi transplanted mice demonstrated improved glucose tolerance by day 17 (Fig. 2j). Collectively, these data suggested that within 4–7 days of iPan administration, increased ALDHhi cells surrounding islets correlated with increased proliferation of insulin+ and insulin− cells, resulting in augmented insulin production and improved endocrine functions.
This study demonstrates the capacity of UCB-derived ALDHhi cells to promote islet regeneration when transplanted directly into the murine pancreas. UCB ALDHhi cells represented a heterogeneous mixture of myeloid cells and haematopoietic/endothelial progenitors readily available for the development of novel cellular therapies for type 1 or type 2 diabetes through the recent establishment of UCB registries for allogeneic transplantation. While i.v. transplantation of UCB ALDHhi cells leads to minimal recovery of islet function, iPan delivery of UCB ALDHhi cells leads to reversal of established hyperglycaemia, increased serum insulin and improved glucose tolerance within 7 days of transplantation. To our knowledge this work represents the first report documenting potent islet recovery after iPan delivery of clinically applicable progenitors isolated from human UCB.
Islet cell proliferation correlated with the presence of UCB ALDHhi cells surrounding damaged islets, and islet number, size and vascularisation were increased 1 month post-transplantation. Although iPan injected ALDHlo cells were occasionally detected in the pancreas at early time points, these cells failed to induce islet-associated proliferation and subsequent recovery of function. Thus, islet regeneration was promoted after timely local exposure to ALDHhi cells, suggesting that islet proliferative or proangiogenic stimuli were provided specifically by iPan injected ALDHhi cells.
Clinical evidence suggests that strategies employing BM or UCB stem cells can potentially benefit diabetic patients via beta cell regenerative or immunomodulatory mechanisms [8, 9]. However, Haller and colleagues recently reported that i.v. infusion of unfractionated UCB cells induced changes in regulatory T lymphocyte frequency but failed to preserve C-peptide . Although our study did not directly address potential immunomodulatory mechanisms, or the activation of putative islet-derived beta cell precursors that may survive STZ toxicity , these experiments establish proof of concept that iPan delivery of purified ALDHhi progenitor cells can formulate a regenerative niche that impacts the behaviour and function of regenerating host islets. Further investigation is warranted to elucidate the molecular pathways activated in signal-receiving beta cells or beta cell precursors during ALDHhi cell-stimulated islet regeneration, as iPan delivery of UCB ALDHhi cells may represent a viable strategy to ‘tip the balance’ in favour of islet expansion vs destruction during diabetes.
We wish to acknowledge H. Broughton for animal care, and i.v. and iPan transplantation, and K. Chadwick and K. Morley for fluorescence activated cell sorting (Robarts Research Institute, University of Western Ontario, London, ON, Canada).
This work was supported by grants from the Canadian Institutes of Health Research (MOP#86702) and a New Investigator Award from the Heart and Stroke Foundation of Canada to D. A. Hess.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
GIB and DAH contributed to the conception and design, analysis and interpretation of data, and writing and revision of the manuscript. DMP and JMH-L contributed to the analysis and interpretation of data, and revising the manuscript critically for important intellectual content. All authors approved the final manuscript. All authors are affiliated with the Krembil Centre for Stem Cell Biology, Robarts Research Institute, Vascular Biology Group, Department of Physiology and Pharmacology, University of Western Ontario.