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

Abstract

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 b 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 ALDHactivity 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% 6 7.0% CD34 1 /CD38 2 cells, 37.0% 6 6.9% CD34 1 /CD133 1 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 isletassociated 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 SIGNIFICANCE STATEMENTAlthough fresh umbilical cord blood ALDHhi cells represent a promising population for the stimulation of islet regeneration, the low number of ALDHhi cells in UCB limits widespread application in diabetic patients. Clinical use of UCB ALDHhi cells to treat diabetes will require expansion without compromising previously reported islet regenerative functions. In order to generate more ALDHhi cells for regenerative applications, we developed a clinically applicable culture protocol that resulted in a significant increase in ALDHhi cells in 6 days. High ALDHexpression was diminished as culture time progressed. Therefore, we re-selected expanded progeny using ALDH-activity after culture, and compared the islet regenerative function of more differentiated (ALDHlo) versus less differentiated (ALDHhi) subsets. Compared to expanded progeny with low ALDH activity, the ALDHhi cell subset more highly expressed primitive cell surface markers, and demonstrated a pro-angiogenic transcription signature and enhanced hematopoietic colony formation in vi...

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

“…Detailed Methods have been provided in Supporting Information Materials that describe routine, previously published procedures in our laboratory such as UCB ALDH hi cell isolation , flow cytometric characterization of cell surface phenotype , and hematopoietic progenitor colony formation . Also included in Supporting Information Methods is detailed descriptions of transplantation experiments performed with immunodeficient mice after FAL surgery , analyses of limb perfusion using laser Doppler perfusion imaging , gait analyses by Catwalk imaging , and immunohistochemical analyses used to characterize vessel regeneration and human cell engraftment in ischemic muscle .…”

Concise Review: Cell Therapy for Critical Limb Ischemia: An Integrated Review of Preclinical and Clinical Studies

“…CD34 + cell selection seemed to be a promising approach in the ACT34‐CLI (Autologous Cell therapy using CD34 cells for CLI) trial performed by Losordo et al, showing that IM transfer of G‐CSF‐mobilized CD34+ cells at high dose trended toward lower amputation incidence ( p = .054) compared with placebo in a small cohort of patients . A similar approach used selection based on high ALDH activity, a protective oxidizing enzyme and highly expressed in progenitor cells of multiple mesodermal lineages . Notably, ALDH hi cells highly coexpressed CD34, and accelerated recovery of perfusion in NOD/SCID mice with femoral artery ligation .…”

“…However, extended culture is known to negatively impact regenerative function. Using the established principle that ALDH‐expression decreases as cells mature , we have used high ALDH‐activity to reselect subsets from expanded lineages with enhanced pro‐vascular functions . A similar strategy can be employed using cell surface marker expression for CD34, CD133, and CD146, representing HPC, EPC, and MSC, respectively.…”

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

“…D, E). Collectively, UCB ALDH hi cells could be expanded up to 6 days and retain the capacity to improve perfusion after transplantation . Extending culture to 9 days resulted in loss of vascular regenerative function.…”

“…CD31+ cells were counted in a blinded fashion from nine fields per section. Immunofluorescence microscopy was used to visualize proliferating (EdU + ) CD31+ cells as previously described .…”