**5. Aldefluor activity in stem cells and cancer stem cells**

ALDHbr (ALDH-bright) cells can be detected with ALDEFLUOR reagent by using flow cytometry or fluorescent microscopy. These ALDHbr cell populations are isolated from adult tissues by flow sorting.

Aldehyde Dehydrogenase: Cancer and Stem Cells 19

Therefore, recently the importance of ALDH activity in normal and malignant stem cell functions, and the potential diagnostic and therapeutic implications gain importance

**Figure 1.** The Aldefluor® Assay. Firstly, ALDH positive cell will uptake BODIPY-aminoacetaldehyde by passive diffusion and then convert BODIPY-aminoacetaldehyde into BODIPY-aminoacetate. Then BAA is retained inside cells, causing the subset of ALDHhi cells to become highly fluorescent (Marcato*,* 

Until today, studies showed that BM ALDHbr populations may be useful in several cell therapy applications (Gentry*, et al.*, 2007)**.** According to this information, ALDHbr population

**7. Aldehyde dehydrogenase in regenerative medicine** 

(Moreb, 2008).

*et al.*, 2011).

ALDH activity was shown in human and mouse bone marrow hematopoietic progenitor cells (HPCs) by Jones et al. for the first time (Jones*, et al.*, 1995). ALDH was assayed by using a new substrate with low light scatter properties with flow cytometry. Now this method was improved and known as Aldefluor assay. Aldefluor assay can be used for to measure ALDH activity of adult tissue cells, primary cancer cells and cultured cells. Aldefluor assay is based on the conversion of fluorescent non-toxic substrate for ALDH substrate to the fluorescent reaction product. Non-toxic substrate for ALDH freely diffuses into intact and viable cells. The BODIPY aminoacetaldehyde is converted to the fluorescent product BODIPY aminoacetate by ALDH activity (Figure 1). In this assay, a specific inhibitor of this reaction (diethylaminobenzaldehyde-DEAB) is used to control for background fluorescence. Aldehyde dehydrogenase plays a role as a cancer stem cell marker comes down to the specific isoform.

Stem and progenitor cells are identified as cells with low side scatter and high expression of ALDH. DEAB allows to distinguish between ALDH-bright cells and cells with low ALDH activity. Generally, 105-106 cells are suspended in Aldefluor assay buffer containing BODIPY aminoacetaldehyde with/without DEAB. Aldefluor was excited at 488 nm and fluorescence emission was detected at 530/30 (van den Hoogen*, et al.*, 2010). This assay provides a successful isolation of viable HSCs and more recently ALDH positive CSCs. However, aldefluor assay detects the ALDH activity of several ALDH isoforms expressed in the cells. ALDH1A1 is not the only isoform responsible from aldeflour activity. In some studies, it was demonstrated that ALDH1A1-deficient hematopoietic cells showed aldefluor activity owing to ALDH2, ALDH3A1 and ALDH9A1 isoforms (Marcato*, et al.*, 2011).
