Innovita Research Foundation

I.R.F. / Aging news / General / 11013102

Expansion of Hematopoietic Stem Cells for Clinical Applications
Posted on: January 31, 2011

Cord blood (CB) hematopoietic stem cell (HSC) transplantation plays a growing role in the treatment of a wide variety of malignant and non-malignant disorders such as leukemia, lymphoma, lymphoproliferative disorders and bone marrow failures. Cord blood, as a source of HSCs, widens the pool of potential donors compared to bone marrow and peripheral blood stem cells due to its ease of harvest, availability, less stringent HLA matching criteria and lower graft-versus-host disease. However, despite the advantages, the number of CB HSC transplantations recorded in a 2008 survey in Europe is only 7% of the total allogeneic HSC transplantations. This is due to the low number of cells collected per unit of CB that restricts its use to children and lightweight recipients. This cell dose limitation leads to a lower success rate in adult recipients, marked by a delay in engraftment and vulnerability to infectious morbidity.

In order to address cell dose limitations, differing strategies to expand CB HSCs ex vivo have been proposed. Importantly, ex vivo expansion is geared not only to increase the number of transplanted cells, but the number of lineage-committed progenitor cells that can accelerate the engraftment process and reduce the risk of infection. Thus far, only one study has shown a marked improvement in the engraftment rate of ex vivo expanded CB HSC in a phase 1 clinical trial.

One of the attempts to improve HSC expansion ex vivo includes the incorporation of stromal components in culture to recreate the hematopoietic microenvironment in which stroma-derived extracellular matrix (ECM) and stem cells provide complex molecular cues to support hematopoiesis. Up to two decades ago, it was believed that direct physical contact between HSC and stromal components was required for HSC maintenance. However, more recent studies show that stromal cell-derived conditioned media, in which various cytokines and proteoglycans are found, is sufficient to maintain HSCs. Furthermore, the complex role of glycosaminglycans (GAG) in supporting hematopoiesis, particularly those GAGs that originate from marrow-derived proteoglycans, is increasingly being elucidated; GAG can bind and regulate the distribution of various hematopoietic cytokines and maintain long-term culture-initiating cells (LTC-IC). The positive effect of marrow-derived GAGs toward LTC- IC maintenance is attributed to heparan sulfate (HS) that is present as a minor component (10 to 12%) of the total GAG in the stroma-conditioned media.

Heparan sulfate has been long known to facilitate various physiological functions through its interaction with proteins. Scientists have shown temporal changes in HS proteoglycan profiles during osteogenesis. Moreover, the isolation, purification and subsequent reintroduction of these HSs from different stages of growth and osteogenic differentiation can differentially regulate cell growth. They have also shown that purified HS with affinity for FGF-2 can sufficiently support mesenchymal stem cell (MSC) proliferation in the absence of exogenously added FGF-2, presumably by supporting the activity of endogenous FGF-2. The use of HS instead of FGF-2 for MSC expansion proved to be more beneficial, since the continuous use of exogenous FGF-2 during MSC osteogenic differentiation inhibits mineralization.

In the current work, scientists explored the effect of HS derived from HSC-supportive human bone marrow stromal cells towards the expansion of human CB HSCs and how it compares to the effect of total GAG. They hypothesize that human bone marrow-derived HS, herein referred to as HS5, can better maintain and enhance the proliferation of CB HSC in the presence of hematopoietic cytokines compared to total GAG. Total GAG and HS5 were isolated from the human bone marrow stromal cell line HS-5, herein referred to as BMS5, as this cell line has been shown to support the growth of hematopoietic stem cells both in co-culture systems and in the presence of its conditioned media. They examined the effect of total GAG or HS5 toward the expansion of purified CD34+ cells from human CB units and CD34+ CD38- subpopulation in the presence of a combination of hematopoietic cytokines: stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt-3L) and thrombopoietin (TPO). Hematopoietic stem cell phenotypic marker expression and the formation of myeloid-colony-forming cells were assessed to reveal the pluripotency and state of differentiation of the cells.

The effect of HS5 toward CB HSC expansion was also compared to heparin derived from porcine mucosa, an HSC non-supportive cell source. Heparin shares identical disaccharide unit building blocks with HS, though is more extensively sulfated compared to the latter and, hence, binds a greater number of cytokines than HS. It was shown that HS at low dose, but neither the total GAG nor heparin, has the ability to increase the number of myeloid lineage- committed progenitor cells despite having no effect towards overall expansion of HSCs and the more primitive, quiescent CD34+ CD38- cell population. Thus, bone marrow-derived HS may have the potential to accelerate engraftment time by facilitating the expansion of committed cells from CB HSCs.

Source: Bramono DS, Rider DA, Murali S, Nurcombe V, Cool SM.; The Effect of Human Bone Marrow Stroma-Derived Heparan Sulfate on the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells.; Pharm Res. 2010 Dec 24. [Epub ahead of print]
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