FACS purification of multiple human progenitor subtypes using ALDH

mesenchymal lineages134,188_{. Purification of BM or UCB cells based on high ALDH-} activity can be used to simultaneously isolate adult stem and progenitor cell subtypes for the preclinical development of regenerative therapies inducing tissue repair. Focusing on transplantation studies using human ALDH-expressing progenitor cells for hematopoietic reconstitution, blood vessel formation, and islet regeneration in immunodeficient mice, the Hess laboratory aims to understand how the multiple progenitor subtypes act together to formulate a regenerative niche and to coordinate complex regenerative processes.

1.4.1 FACS purification of multiple human progenitor subtypes using

ALDH activity

Intracellular ALDH activity can be quantified using a fluorescent substrate for ALDH, termed Aldefluor reagent 186

. First synthesized by Clayton Smith’s group in 1999, Aldefluor reagent is a Bodipy fluorochrome conjugated to an aminoacetaldehyde

molecule, an uncharged moiety that can freely cross through the cell membrane. Once inside the cell, cytoplasmic ALDH1A1 converts Aldefluor into a metabolized by ALDH into an anion that becomes trapped in the cell due to its negative charge. Under pharmacological inhibition of ABC transporters contained within Aldefluor buffer, cells with high ALDH activity retain Aldefluor substrate and fluoresce brightly, while cells with lower ALDH activity are more dimly fluorescent. Thus, high-speed fluorescence- activated cell sorting (FACS) can efficiently purify UCB or BM MNC with low side scatter and low versus high ALDH activity. The integrity and function of the isolated cells are not compromised by this procedure since upon removal of the Aldefluor buffer, ATP-binding cassette transporters become reactivated, and Aldefluor is actively effluxed, returning the cell to its original state. Thus, the Aldefluor purification procedure is clinically applicable for the efficient sorting of multiple functional human progenitor cell types based on a highly conserved stem cell function. The amount of ALDH activity in all viable cells falls along a spectrum from low ALDH activity (ALDHlo

) to high ALDH activity (ALDHhi

), where ALDHlo

versus ALDHhi

cells are distinguished by cluster gating using diethylaminobenzaldehyde or DEAB, a pharmacological inhibitor of ALDH1A1. The basic premise of the Aldefluor assay to assess ALDH activity with FACS is summarised in Figure 1.1.

It has previously been shown that purified ALDHhi

cells from BM and UCB highly co- expressed stem cell-associated surface markers (CD34, CD133, c-kit) and were enriched for multipotent hematopoietic and mesenchymal stromal progenitors, as well as precursor cells with endothelial colony forming cell (ECFC) capacity in vitro134,156,188,189

. In contrast, ALDHlo _{cells were primarily comprised of mature leukocytes (primarily T- and B-cells)} and demonstrated little progenitor function in vitro134

. Therefore, high ALDH activity simultaneously purifies multiple progenitor cell subtypes ideal for lineage-specific expansion in vitro. Subsequently, purified ALDH-purified mixed progenitor cells or their ex vivo expanded progeny can be assayed for regenerative functions after xenotransplantation into a variety of immunodeficient models of tissue damage.

Figure 1.1. Schematic overview of FACS isolation of ALDHhi

cells using Aldefluor. (A) Aldefluor passively diffuses into cells where is metabolized by ALDH into an anion which can no longer diffuse out of the cell, this leads to accumulation of a fluorescent signal that can be detected and selected by FACS. (B) DEAB inhibition of ALDH activity identifies ALDHlo

population for gating. (C) Aldefluor labeling allows efficient selection of ALDHhi

1.4.2 Cells with high ALDH activity possess hematopoietic

repopulating capacity

Hematopoietic stem cells (HSC) and lineage-specific hematopoietic progenitors are responsible for the replenishment and maintenance of blood after BM transplantation190,191

. These cells can be isolated from BM, cytokine-mobilized peripheral blood, or UCB based on expression of the cell surface markers CD34 and CD133, and assayed for hematopoietic repopulating function after transplantation into sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice192-194

. Storms et al. first established that the ALDHhi

fraction of human UCB was enriched for primitive hematopoietic progenitors in vitro and was depleted of lineage-committed hematopoietic cells186_{. Subsequently, prospective lineage depletion in combination with} the commercially available Aldefluor reagent (Stemcell Technologies, Vancouver, Canada) was used by our group to demonstrate that transplantation of human UCB ALDHhi

cells into NOD/SCID mice resulted in multilineage human hematopoietic engraftment156

. Greater than 70% of the UCB ALDHhi

cell population co-expressed the HSC-associated cell surface markers CD34 and CD133156

. ALDHhi

CD34+

cells were highly enriched for short-term myeloid progenitors while ALDHhi

CD34− cells represented precursors to the CD34+

population that also demonstrated NOD/SCID- repopulating cell (SRC) capacity195-197

.

Our laboratory has also demonstrated that while both ALDHhi

CD133−

and ALDHhi CD133+

cells demonstrated clonogenic hematopoietic progenitor function in vitro, only the ALDHhi _CD133+ _{population was able to engraft the murine BM after intravenous} injection198_{. Furthermore, prospective selection based on both high ALDH activity and} CD133 increased the frequency of SRC by tenfold compared to selection by CD133 alone. Notably, ALDHhi

CD133+

cells demonstrated enhanced hematopoietic repopulating function in serial secondary transplants while maintaining primitive hematopoietic phenotypes (CD34+

CD38−

)198

. In addition to long-term hematopoietic repopulating function, human UCB ALDHhi

CD133+

cells also showed previously unrecognized engraftment in nonhematopoietic tissues such as the liver, lung, heart,

brain, pancreas using the highly sensitive human cell-tracking NOD/ SCID MPSVII model199

.

Later studies established that hematopoietic engraftment after human UCB transplantation in immunodeficient mice occurs faster with increasing ALDHhi

cell doses 200

. Similar to CD34 expression, clinical reconstitution rates following transplantation of BM or mobilized peripheral blood can be directly correlated with the number of ALDHhi cells infused201,202

. As a result of these promising preclinical data and direct potential for clinical translation, recently completed clinical trials designed to assess the safety and efficacy of transplanted allogeneic human UCB ALDHhi

cells to enhance the rate of engraftment in the treatment of haematological dysfunction (www.clinicaltrials.gov trial no. NCT00692926) have been reported. In summary, high ALDH activity is now well established as a functional characteristic of repopulating hematopoietic cells, and ALDH activity appears to be a superior indicator of the quality of BM or UCB samples for transplantation compared to standardized CD34+

counts201 .