Development of a perfusion bioreactor for culturing hematopoietic stem cells

Project Associates / Assistants
T Madhusudhan

The project aims at development of a bioreactor and its operation for cultivation of hematopoietic stem cells, mimicking bone marrow microenvironment. The specific objectives are (i) development of a biocompatible 3-D matrix from natural and/or synthetic polymers, (ii) culture of murine HSC on 3-D matrix, pre-exposed to either stromal cells or extra cellular matrix (ECM) components, (iii) study on grafting of ex vivo expanded stem cells, (iv) study on self-propagating potential of early progenitor stem cells and its differentiation into myeloid lineage, and (v) development of a prototype bioreactor for expansion of early progenitor stem cells and optimization of bioreactor operating parameters.

Ex vivo expansion of murine stem cells are initiated in preformed stroma, as well as on extra cellular matrix (ECM) coated surface. The idea of using ECM components of bone marrow, such as, collagen-1, fibronectin, and laminin coated surface is to study the interaction of stem cells with these ECM and few key cytokines, such as, stem cell factor (SCF), Flt-3 ligand, and IL-3 for it’s self-renewal and differentiation. It has been found that IL-3 alone expands murine progenitor stem cells on both fibronectin and laminin support, with greater retention of myeloid colony forming cells in laminin support. These preliminary results possibly implicate the differential roles of integrin receptors (a₄b_1, a₅b_1, and a₆b₁) in the maturation of myeloid cells and maintaining short-term bone marrow repopulating ability.

Inside bone, hematopoietic cells grow, mature, and function like a tissue, so in marrow cellular arrangement is expected to be three-dimensional. Supporting to this notion, a successful culture of HSC is thus possible on recreating bone marrow microenvironment: by providing ECMs and growth factors, supplying nutrients and finally by inducing tissue like growth in a three-dimensional support. During the course of this investigation a prototype hand woven three-dimensional matrix was made from polyethylene terepthalate (PET) film and fiber. PET has been found as biocompatible as polystyrene used in tissue culture flask. First, we addressed the question whether HSCs grow on three-dimensional support are functionally better than that grow on two-dimensionally. We compared two- and three-dimensional culture systems in terms of colony forming unit-cell (CFU-C) and cobblestone area forming cell (CAFC) assays, and presence of Sca-1 cell surface marker. Our preliminary results indicated that the functional properties of HSCs are better preserved if grown on three-dimensional support.

No suitable model has been reported for studying in vitro homing of primitive engraftable stem cells and its interaction with BM stroma cells. An In vitro model is necessary to study the role of cell surface adhesion molecules and ECM components on the interaction of stroma and engraftable progenitor cells. Nevertheless, manipulated progenitor stem cells, whether they are expanded in presence of cytokines, or they carry heterologous gene(s) requires to be tested in vitro before they are evaluated in animal model. An in vitro model for homing of stem cells on pre-established stroma layer has been established. The stroma-adhered cells were found to engraft, multiply and differentiate in BM of age-matched mice. In vitro study revealed that initially the adhesion of BM cells on irradiated stroma was increased with time and attained a peak at 2 h of contacts. During that time 44.1 ± 6.5 % (n=8) cells were adhered, and this value was maintained up to 6 x 10⁶ cells. The adhered cell fraction was enriched by 3.9-, 2.5-, and 1.7-fold Sca-1, CFC, and CAFC, respectively, as compare to the fresh BM cells. This adhered cells homed to BM with engraftment efficiency of 11.8 ± 2.5 % (n=6). The homed cells reconstitute BM of myeloablative mice by self-renewing and differentiating into myeloid cells.

Publications

Original peer-reviewed articles

1.     Madhusudhan T, Richhariya A, Mazumder S and Mukhopadhyay A. (2003) An in vitro model for grafting of hematopoietic stem cells predicts bone marrow reconstitution of a myeloablative mice. J Hematother Stem Cell Res (in press).

2.     ¶Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M and Aggarwal BB (2002) Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation. Oncogene 21:8852‑8861 (¶on deputation/work done elsewhere).

3.    ¶Mukhopadhyay A, Shishodia S, Suttles J, Brittingham K, Lamothe B, Nimmanapalli R, Bhalla KN and Aggarwal BB (2002) Ectopic expression of protein tyrosine kinase Bcr-Abl suppresses TNF-induced NF-kB activation and IkBa phosphorylation: Relationship with downregulation of TNF receptors. J Biol Chem 277:30622-30628 (¶on deputation/work done elsewhere).

4.    ¶Anto RJ, Mukhopadhyay A, Shishodia S and Aggarwal BB (2002) Cigarette smoke condensate activates nuclear transcription factor-kB through phosphorylation and degradation of IkBa: correlation with induction of cyclooxygenase-2. Carcinogenesis 23:1511-1516 (¶on deputation/work done elsewhere).

5.    ¶Bosshart H, Mukhopadhyay A, Fiumara P, Li Y, Darnay BG, Aggarwal BB and Younes A (2002) Expression of survival receptors in Hodgkin disease cell lines. Blood 99:3484-3486 (¶on deputation/work done elsewhere).

Reviews/Proceedings

1.    Mukhopadhyay A, Madhusudhan T and Kumar R (2003) Hematopoietic stem cells: Clinical requirements and developments in ex vivo culture. In: Advances in biochemical engineering/biotechnology (Ed: Scheper T) Springer-Verlag, Germany (in press).