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Ex-vivo Expansion of Human Hematopoietic Stem Cells
Ando K et al, Blood 2006;107:3371
Summary by: Nirmala Hariharan, Shweta Rane

LAY SUMMARY 

Stem cells are immature or undifferentiated cells that have the ability to generate an identical cell, a process known as ‘self-renewal’ and also form any type of cell in the body. Hematopoietic stem cells (HSCs) are the prototypical stem cells found in the bone marrow that are capable of producing all lineages of white and red blood cells such as lymphocytes, granulocytes, platelets, erythrocytes, macrophages, monocytes, etc. by a process known as ‘hematopoiesis’. HSCs have gained importance in medical research over the years to treat blood related diseases and cancer and extensive studies are being conducted to analyze their ability of expansion ex-vivo (outside the body).  

Techniques such as the use of severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have been used in the past to characterize HSCs in culture. A SCID mouse has a defective immune system which lacks white blood cells (WBCs) to fight infection. HSCs given to such mice help repopulate the WBCs and in turn assist in infection resistance. Such cells are called SCID Repopulating Cells (SRC) and the technique is called SRC assay. This study have compared different techniques to identify the most reliable method for ex vivo expansion of HSCs. Limiting dilution analysis (LDA) is considered “the gold standard” for measuring expansion, but being a statistical method, it does not directly measure self renewal ability of the cell. The current study proposes a technique using retroviral (virus which has RNA instead of DNA as its genetic material) insertion and uses two different methods of transplantation of foreign cells (human) in a mouse: direct intra-bone marrow transplantation (iBMT) and intra-venous injection of the HSCs.  

Based on the results obtained from the study, the authors conclude that the HSCs in culture can undergo two types of cell division: symmetric and asymmetric. When the two daughter cells formed are identical to the parent cell then it is ‘symmetric cell division’ and when one of the daughter cell resembles the parent cell whereas the other daughter cell is a differentiated cell, its is ‘asymmetric cell division’. Further studies are in progress to identify the differences between the two HSC classes

SCIENTIFIC SUMMARY 

This study addresses a major problem, namely ex-vivo expansion of human Hematopoietic stem cells (hHSCs). Experimental techniques were done to characterize HSCs using severe combined immunodeficient (SCID) mice in studies to understand their repopulating ability (SRC assay). SRC assay determines the in vivo potential of reconstitution or repopulation of the SRC cells in a SCID mouse. This study have compared different techniques to identify the most reliable method for ex-vivo expansion of HSCs. Limiting dilution analysis (LDA), a current method used to quantitatively analyze stem cell expansion is considered “the gold standard”, but being a statistical method it does not directly measure the self-renewal ability of the HSCs.  

The current study uses CD34+ hHSCs from umbilical cord blood (Relative SRC: 3-4) from full term deliveries. These cells are isolated and expanded using sera-free and stroma dependant (SFSD) culture system. The HSCs were labeled with 5-and 6-carboxyfluorescein diacetate succinimidyl ester (CSFE), which is a fluorescence marker to track the cells. To study clonal analyses, the authors used retroviral gene insertion in the CD34+/EGFP+ cells. The clones are then transplanted in NOD/SCID mice using two well known xenotransplantation techniques: SRC assay by intra-venous injection via the tail vein and intra bone marrow transplantation (iBMT). The clones with unique genomic-proviral junction sequence were identified using linear amplification mediated-PCR (LAM-PCR). Fluorescence associated cell sorting (FACS) is used to analyze clonal kinetics.  

To compare the differences in SRC frequencies between cultured and uncultured CD34+ cells a quantitative SRC assay was used. CD34+ cells were found to undergo 9.8 fold increases in SRC expansion following sera-free stromal dependent (SFSD) culture system (Figure 1). Since SRC assay involves intravenous injection of CD34+ cells, the homing and proliferative abilities of the cells should be taken into consideration. Similarly, a comparison of lentivirus (retrovirus) infected SRCs with and without SFSD culture system was carried out. The infected cells were transplanted by intra-venous injections and iBMT in separate experiments. The SFSD cultured cells showed more efficiency for forming lineage committed cells expressing CD19+ (lymphoid) and CD33+ (myeloid) markers. LAM-PCR was conducted to identify and select each clone within the mice. Secondary transplantation experiments were designed to analyze the self renewal ability of each clone based on the expression of lymphomyeloid differentiation markers in the primary and secondary mice. These experiments helped prove that hHSCs can be cultured in vitro.  

Conclusion:

Based on the results obtained from the study using long-term repopulating cells (LTRCs), the authors concluded that there are two classes of hHSCs in culture: one which expands by undergoing symmetric self-renewal division at some stage during SFSD and the other that undergoes asymmetric self-renewal division resulting in HSC maintenance (Figure 2 represents a typical stromal niche). Further studies need to be conducted to gain an in depth knowledge of the differences in these two classes (Figure 3). The method of clonal analysis presented in this study claims to show promising results for ex vivo expansion of HSCs in a clinically approved manner. 

Comment:

The results leave a question of whether the starting populations are all HSCs or if they include progenitors. This concern is valid since HSCs have long dividing times and show low efficiency in retroviral insertion.