**2. Hematopoietic stem cells**

Hematopoietic stem cells are a well characterized stem cell type which has been used in bone marrow transplantation for treatment of hematological malignancies as well as nonmalignant disorders (Warner et al, 2004). In fact, bone marrow (BM) has been, for many years, transplanted as an unfractionated cell pool, until researchers discovered which cellular components were responsible for the engraftment of the donor hematopoietic and immune systems in marrow-ablated patients.

HSCs present self-renewal potential and differentiation capacity into blood lineages. The self-renewal concept means that when stem cells divide, 50% of the daughter cells, on average, is committed with a cell lineage; the remaining 50% do not differentiate; therefore

Hematopoietic Stem Cell in Acute Myeloid Leukemia Development 263

Fig. 2. HSCs differentiation pathways. HSCs could differentiate into specific lymphoid and myeloid cell types. Common lymphoid progenitors (CLPs) can differentiate into all types of lymphocytes and common myeloid progenitors (CMPs) can give rise to all classes of myeloid cells (megakaryocytes, erythrocytes, granulocytes and macrophages) (Adapted

Morphologically, hematopoietic stem cells are undifferentiated and resemble small lymphocytes. Normally, a large fraction is quiescent, in the G0 phase of the cell cycle, which protects them from the action of cell cycle-dependent drugs. The quiescent state of stem cells is maintained by transforming growth factor-β (TGF-β). The activity of TGF-β is mediated by p53, a tumor suppressor gene that regulates cell proliferation and targets the cyclindependent kinase inhibitor p21 (Gordon, 2005). Quiescence of HSCs is critical not only for protecting the stem cell compartment and sustaining stem cell pools during long periods of

Quiescence regulation in HSCs is also of great importance for understanding the pathophysiological origins of many related disorders. Interestingly, many of the intrinsic transcriptional factors that maintain HSCs quiescence are found to be associated with leukemias. For example, chromosomal translocations resulting in the fusion of FoxOs and myeloid/lymphoid or mixed lineage leukemia have been reported in acute myeloid leukemias.

time, but also by minimizing the accumulation of replication-associated mutations.

from Du et al., 2008).

**2.1 Characterization** 

the process maintains the same number of stem cells. This is accomplished by the so-called asymmetric cell division, so that each dividing stem cell originates one new stem cell and one differentiated cell **(**Gordon, 2005) (Figure 1). In the symmetric division, the stem cells originate 100% of identical stem cells.

Fig. 1. Schematic illustration of two different types of HSCs division. On the left, the symmetric division in which mitosis originates two identical stem cell daughters. On the right, the characteristic stem cells asymmetric division where each dividing stem cell forms one new stem cell and one differentiated cell.

HSCs are classified as multipotent stem cells due to their ability to differentiate in lymphoid as well as myeloid cells types; however, some studies showed that transplanted bone marrow cells can contribute to the repair and regeneration of a spectrum of other tissue cell types including those from brain, muscle, lung and liver.

Lymphoid cell lineage includes T and B cells, while megakaryocytes, erythrocytes, granulocytes and macrophages belong to the myeloid lineage. These two lineages derive from different progenitor cells. Common lymphoid progenitors (CLPs) can differentiate into all types of lymphocytes without noticeable myeloid potential under physiological conditions. Similarly, common myeloid progenitors (CMPs) can give rise to all classes of myeloid cells with no or extensively low levels of B-cell potential (Kondo, 2010). It is likely that differences in the expression levels of transcription factors determine the lineage affiliation of a differentiating cell (Figure 2). The transcription factors PU.1 and GATA-1 have been implicated in myeloid and erythroid/megakaryocyte lineage differentiation, respectively (Gordon, 2005).

the process maintains the same number of stem cells. This is accomplished by the so-called asymmetric cell division, so that each dividing stem cell originates one new stem cell and one differentiated cell **(**Gordon, 2005) (Figure 1). In the symmetric division, the stem cells

Fig. 1. Schematic illustration of two different types of HSCs division. On the left, the symmetric division in which mitosis originates two identical stem cell daughters. On the right, the characteristic stem cells asymmetric division where each dividing stem cell forms

HSCs are classified as multipotent stem cells due to their ability to differentiate in lymphoid as well as myeloid cells types; however, some studies showed that transplanted bone marrow cells can contribute to the repair and regeneration of a spectrum of other tissue cell

Lymphoid cell lineage includes T and B cells, while megakaryocytes, erythrocytes, granulocytes and macrophages belong to the myeloid lineage. These two lineages derive from different progenitor cells. Common lymphoid progenitors (CLPs) can differentiate into all types of lymphocytes without noticeable myeloid potential under physiological conditions. Similarly, common myeloid progenitors (CMPs) can give rise to all classes of myeloid cells with no or extensively low levels of B-cell potential (Kondo, 2010). It is likely that differences in the expression levels of transcription factors determine the lineage affiliation of a differentiating cell (Figure 2). The transcription factors PU.1 and GATA-1 have been implicated in myeloid and erythroid/megakaryocyte lineage differentiation,

originate 100% of identical stem cells.

one new stem cell and one differentiated cell.

respectively (Gordon, 2005).

types including those from brain, muscle, lung and liver.

Fig. 2. HSCs differentiation pathways. HSCs could differentiate into specific lymphoid and myeloid cell types. Common lymphoid progenitors (CLPs) can differentiate into all types of lymphocytes and common myeloid progenitors (CMPs) can give rise to all classes of myeloid cells (megakaryocytes, erythrocytes, granulocytes and macrophages) (Adapted from Du et al., 2008).

#### **2.1 Characterization**

Morphologically, hematopoietic stem cells are undifferentiated and resemble small lymphocytes. Normally, a large fraction is quiescent, in the G0 phase of the cell cycle, which protects them from the action of cell cycle-dependent drugs. The quiescent state of stem cells is maintained by transforming growth factor-β (TGF-β). The activity of TGF-β is mediated by p53, a tumor suppressor gene that regulates cell proliferation and targets the cyclindependent kinase inhibitor p21 (Gordon, 2005). Quiescence of HSCs is critical not only for protecting the stem cell compartment and sustaining stem cell pools during long periods of time, but also by minimizing the accumulation of replication-associated mutations.

Quiescence regulation in HSCs is also of great importance for understanding the pathophysiological origins of many related disorders. Interestingly, many of the intrinsic transcriptional factors that maintain HSCs quiescence are found to be associated with leukemias. For example, chromosomal translocations resulting in the fusion of FoxOs and myeloid/lymphoid or mixed lineage leukemia have been reported in acute myeloid leukemias.

Hematopoietic Stem Cell in Acute Myeloid Leukemia Development 265

According to its hematopoietic repopulation capacity, the hematopoietic stem cell pool can

a. short-term HSCs, capable of generating clones of differentiating cells for only 4–6

b. intermediate-term HSCs, capable of sustaining a differentiating cell progeny for 6–8

HSCs can be harvested from healthy donors either by bone marrow aspiration, peripheral stem cell mobilization or from umbilical cord blood (Dick, 2003). HSCs located in the bone marrow present an estimated frequency of 0.01% of total nucleated cells and can be collected by iliac crest puncture and then separated from the other blood cells by magnetic beads or

Umbilical cord blood (UCB) is a source of the rare but precious primitive HSCs and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders treated with myeloablative therapy. UCB cells possess an enhanced progenitor cell proliferation capacity and self-renewal *in vitro*. UCB is usually discarded and it exists in almost limitless supply. The blood remaining in the delivered placenta is safely and easily collected and stored. The predominant collection procedure currently involves a relatively simple venipuncture, followed by gravity drainage into a standard sterile anti-coagulant-filled blood bag, using a closed system, similar to that

Peripheral blood hematopoietic stem cells (PBSCs) have numerous advantages in comparison with traditionally used bone marrow. PBSCs collection by leukapheresis procedure is simple and better tolerated than bone marrow harvest. PBCSs are mobilized by myelosupressive chemotherapy or/and hematopoietic growth factors. Leukapheresis product contains PBSCs along with committed lineage of progenitors and precursors which

Unfortunately, the expansion of HSCs *in vitro* is difficult to achieve because the proliferation is accompanied by differentiation. This is presumably caused by a lack of appropriate cues that are provided *in vivo* by the microenvironment. The most excellent defined culture medium for HSCs expansion is supplemented with cytokines such as fetal liver tyrosine kinase-3 ligand (FLT3-L), stem cell factor (SCF), interleukin-3 (IL-3) and thrombopoietin (TPO). Interestingly, mesenchymal stem cells (MSCs), which are characterized by multi-differentiation potential, are important players of the bone marrow HSCs niche. In recent years, MSCs have been shown to

Hematopoiesis is a highly coordinated process wherein HSCs differentiate into mature blood cells supported by a physical environment called **niche** (Figure 3). The bone marrow

utilized for whole blood collection (Bojanic & Golubic Cepulic, 2006).

support HSCs maintenance and engraftment (Jing et al., 2010).

c. long-term HSCs, capable of maintaining hematopoiesis indefinitely (Testa, 2011).

**2.2 Classification** 

weeks;

**2.3 HSC sources** 

cell sorting.

be subdivided into three main groups:

months before becoming extinct;

contribute to faster hematopoietic recovery.

**3. Factors involved in hematopoiesis** 

The majority of normal HSCs are present among the CD34+/CD38− bone marrow cell fractions; some HSCs are also observed among CD34−/Lin− cells; CD34+/CD38+ cell fractions contain some HSCs but endowed with short-term repopulating activity. Other recognized marker is the tyrosine kinase receptor c-kit (CD117), concomitantly with the lack of terminal differentiation markers (as CD4 and CD8; Figure 3) (Rossi et al., 2011).

Primitive HSCs populations show low fluorescence ratios after Hoechst 33342 and Rhodamine 123 staining; these cells are described as side population (SP). SP cells demonstrate high expression of ATP binding cassette (ABC) transporters as P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2) and lung resistance protein (LRP) (Huls et al., 2009). MDR1 has been implicated in the protection of cells against apoptotic cell death induced by a variety of methods including growth factor deprivation, UV irradiation, ionizing radiation, or tumor necrosis factor-α treatment. BCRP is a halftransporter and characterized as a novel stem cell transporter. Like MDR1, enforced overexpression of BCRP in human MCF-7 breast cancer cells confers a broad spectrum of drug resistance, and elevated levels of expression of BCRP have been reported to be associated with acute myeloid leukemia.

Since ABC transporter function is associated with both normal and aberrant hematopoiesis, it is important to fully characterize the function of this class of transporter proteins in hematopoietic cell differentiation and to define the underlying mechanisms.

Fig. 3. HSCs main surface markers. HSCs express typical antigens as: CD34, CD117, CD164, CD202b, CD31, Flk-1, CD184, CD338 or ABCG2, Notch-1 concomitantly with the lack of terminal differentiation markers (CD4 and CD8).
