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

Introduction

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, ALDH^hi), regenerated the damaged pancreas, after being transplanted into diabetic mice. Interestingly, more mature (differentiated) cells with low ALDH activity did not demonstrate islet regenerative properties^2-4. The diabetic mice that were transplanted with ALDH^hi UCB had lower blood glucose levels, and improved glucose tolerance. However, the number of UCB ALDH^hi cells will not be enough for widespread clinical application in diabetic patients. It is noteworthy that, ALDH^hi UCB cells can be expanded in clinically applicable culture conditions, however the islet regenerative properties of cells expanded from UCB ALDH^hi cells is not known.

Experimental Design

The objective of this investigation was to determine if cells expanded from UCB ALDH^hi 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.     

Results

To test our hypothesis, we first wanted to determine if ALDH^hi UCB can expand in clinically applicable serum-free culture conditions. As it turns out, UCB ALDH^hi 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 ALDH^hi was observed 6 days after culture. Therefore, we used day 6 expanded cells and compared blood progenitor cell surface markers in expanded ALDH^hi and ALDH^lo cells. ALDH^hi cells were enriched blood progenitor cell markers such as CD34, and CD133 compared to ALDH^lo cells. In addition, ALDH^hi cells were able to form more blood colonies compared to ALDH^lo cells. These results suggest that, ALDH^hi cells are more primitive progenitor cells, whereas ALDH^lo cells are more differentiated mature cells. We have previously shown that fresh ALDH^hi UCB cells can support the growth of damaged blood vessels in the hind limb, and the pancreas^3-5. Interestingly, culture expanded ALDH^hi 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 ALDH^hi or ALDH^lo cells into our diabetic mouse model^2-4. Interestingly, it was only mice transplanted with culture expanded and purified ALDH^hi cells that showed improved blood glucose levels, and improved glucose tolerance. To understand how expanded ALDH^hi 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 ALDH^hi cells were detected in the pancreas of diabetic mice. Furthermore, only mice transplanted with expanded ALDH^hi cells showed an increase in islet size, islet cell proliferation, and islet vascularization. Therefore, expanded UCB ALDH^hi cells improved islet cell function by augmenting islet cell proliferation and revascularization.