Stem Cells. 2016 Apr;34(4):873-87. doi: 10.1002/stem.2268.
Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions.
Seneviratne AK1,2, Bell GI1,2, Sherman SE1,2, Cooper TT1,2, Putman DM1,2, Hess DA1,2.
- 1Krembil Centre for Stem Cell Biology, Molecular Medicine Research Group, Robarts Research Institute, London, Ontario, Canada.
- 2Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.
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 β 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.
KEYWORDS: Aldehyde dehydrogenase; Diabetes; Hematopoietic progenitor cells; Islet regeneration; Transplantation; Umbilical cord blood
Diabetes is a disease that is associated with high blood glucose levels. Due to growing rates of obesity in an aging population, diabetes can be considered a global health crisis. It is estimated that diabetes currently affects 260 million individuals worldwide, and this number is expected to increase to 360 million by the year 20301. There are two distinct types of diabetes; type-1 diabetes, and type-2 diabetes. In Type-1 diabetes the immune system attacks the b-cells in the islets of the pancreas, which produces insulin, the hormone that facilitates the entry of glucose from the blood to body tissues, where it is utilized for energy. On the other hand, in type 2 diabetes the body tissues are insensitive to insulin, which leads to the dysfunction of β-cells, and ultimately β-cell exhaustion. Thus, β-cell dysfunction is common in both type-1 and type-2 diabetes1.
The symptoms associated with type-1 and type-2 diabetes are currently managed using exogenous insulin, or oral glucose lowering agents. However, these treatments do not have the ability to control glucose levels ‘on demand’, therefore diabetic patients have more extreme peaks and troughs in blood glucose levels. As a result, more than 80% of diabetic patients will develop severe vascular complications including peripheral limb ischemia, heart attack, and stroke. We are attempting to utilize stem cells to correct the β-cell dysfunction in both type-1, and type-2 diabetes.
Stem cells have two defining properties: (1) the ability to renew itself by dividing (self-renew) (2) the ability to give rise to multiple cells in the body (differentiate). Progenitor cells are the progeny of stem cells, and only demonstrate the ability to differentiate. Interestingly, we have previously shown that hematopoietic progenitor cells, that were isolated from human cord blood (UCB), based on high aldehyde dehydrogenase activity (progenitor cell marker, ALDHhi), regenerated the damaged pancreas, after being transplanted into diabetic mice. Interestingly, more mature (differentiated) cells with low ALDH activity did not demonstrate islet regenerative properties2-4. The diabetic mice that were transplanted with ALDHhi UCB had lower blood glucose levels, and improved glucose tolerance. However, the number of UCB ALDHhi cells will not be enough for widespread clinical application in diabetic patients. It is noteworthy that, ALDHhi UCB cells can be expanded in clinically applicable culture conditions, however the islet regenerative properties of cells expanded from UCB ALDHhi cells is not known.
The objective of this investigation was to determine if cells expanded from UCB ALDHhi cells have the ability to stimulate islet regeneration after transplantation into diabetic mice. We hypothesized that only expanded cells that retain high ALDH activity will demonstrate islet regenerative functions.
To test our hypothesis, we first wanted to determine if ALDHhi UCB can expand in clinically applicable serum-free culture conditions. As it turns out, UCB ALDHhi cells expanded by 2-, 20-, 54-fold after 3, 6, and 9 days in culture respectively. We knew that cells become more differentiated when expanded in culture, so we measured ALDH activity of the cell progeny after expansion. As expected, the frequency of cells retaining high ALDH-activity diminished as culture time progressed (30% at day 3, 15% at day 6, <2% at day 9). The highest number of cells that retained ALDHhi was observed 6 days after culture. Therefore, we used day 6 expanded cells and compared blood progenitor cell surface markers in expanded ALDHhi and ALDHlo cells. ALDHhi cells were enriched blood progenitor cell markers such as CD34, and CD133 compared to ALDHlo cells. In addition, ALDHhi cells were able to form more blood colonies compared to ALDHlo cells. These results suggest that, ALDHhi cells are more primitive progenitor cells, whereas ALDHlo cells are more differentiated mature cells. We have previously shown that fresh ALDHhi UCB cells can support the growth of damaged blood vessels in the hind limb, and the pancreas3-5. Interestingly, culture expanded ALDHhi cells highly expressed mRNA for factors such as: FRAS-1-related extracellular matrix protein (frem1), complement factor H (cfh), chordin-like protein 1 (chrdl1), fibrillin-1 (fbn1), angiopoietin-2 (angpt2), semaphorin 4A (sema4a), milk-fat globule-EGF factor 8 (mfge8) that support blood vessel growth. To test the islet regenerative properties of expanded UCB cells, we transplanted saline (vehicle control), expanded bulk cells, or expanded and selected ALDHhi or ALDHlo cells into our diabetic mouse model2-4. Interestingly, it was only mice transplanted with culture expanded and purified ALDHhi cells that showed improved blood glucose levels, and improved glucose tolerance. To understand how expanded ALDHhi cells improved islet function we harvested the pancreas of transplanted mice and measured the presence of transplanted cells, islet size, number, islet cell proliferation, and islet vascular density. Interestingly, only ALDHhi cells were detected in the pancreas of diabetic mice. Furthermore, only mice transplanted with expanded ALDHhi cells showed an increase in islet size, islet cell proliferation, and islet vascularization. Therefore, expanded UCB ALDHhi cells improved islet cell function by augmenting islet cell proliferation and revascularization.
These studies provide proof-of-concept that UCB ALDHhi cells can expand under serum-free hematopoietic conditions, and it is only the culture expanded primitive progenitor population (expanded ALDHhi cells) that retain islet regenerative function after transplantation. Our results, also suggest that expanded ALDHhi cells improve the islet function of diabetic mice by surrounding damaged islets, and secreting factors that support islet blood vessel growth (Figure 1). Although further understanding of the mechanism of how these progenitor cells stimulate islet regeneration is required to maximize islet regeneration in the face of autoimmunity, optimization of culture expansion and re-selection strategies using high ALDH-activity, may ultimately lead to the development of a cell based therapy to treat diabetes.
- Seneviratne, A.K. et al. Expanded Hematopoietic Progenitor Cells Reselected for High Aldehyde Dehydrogenase Activity Demonstrate Islet Regenerative Functions. Stem Cells34, 873-887 (2016).
- Bell, G.I. et al. Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization. Stem Cells Dev21, 97-109 (2012).
- Bell, G.I. et al. Combinatorial human progenitor cell transplantation optimizes islet regeneration through secretion of paracrine factors. Stem Cells Dev21, 1863-1876 (2012).
- Bell, G.I., Putman, D.M., Hughes-Large, J.M. & Hess, D.A. Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration. Diabetologia55, 1755-1760 (2012).
- Putman, D.M., Liu, K.Y., Broughton, H.C., Bell, G.I. & Hess, D.A. Umbilical cord blood-derived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury. Stem Cells30, 2248-2260 (2012).