Ex-vivo manipulation of hematopoietic stem cells (HPCs) has a great potential in clinical applications including gene therapy, gene editing (Project 1), and cell therapy. Identifying systems to permit ex vivo renewal and expansion of pluripotent HPCs is a crucial step in the manipulation of HPCs. In this pilot, we will design, fabricate and evaluate the capacity of nanofiber-matrigel scaffolds with incorporated notch ligand and granulocyte-colony stimulating factor, to permit self-renewal of uncommitted HPCs. The rationale of this design is that nanofiber composition and topography support enhanced cell attachment to the scaffolds, and Notch ligand is sustainably released from the scaffolds to stimulate the HPC growth. The sustained release of Notch ligand will be achieved by the gradual degradation of nanofiber layers and diffusion of Notch ligand from matrigel into the cell culture medium. The layer by layer fabrication of the nanofiber-matrigel scaffolds will be carried out using a standard electrospinning rotating drum apparatus in conjunction with applying a thin layer of matrigel between the nanofiber mesh layers. Degradation rate of the nanofiber layers will be modulated by the choice of the nanofiber composition, and the release rate of Notch ligand will be adjusted by modulating electrospinning parameters.
In Specific Aim 1, multilayer nanofiber-matrigel scaffolds will be fabricated, and optimzed for sustained release of Notch-ligand in the culture medium of hematopoietic progenitor cells.
In Specific Aim 2. The nanofiber-matrigel scaffolds will be evaluated for expansion of the CD34+ hematopoietic stem cell population from cord blood. Our hypothesis is that CD34+ HPC will be expand preferentially on the Notch-ligand sustainably releasing nanofiber scaffolds. Ultimately, these experiments will support Project 1, while also developing possibilities for new avenues of research such as ex vivo expansion of cord blood derived HPCs.
The Notch ligand encapsulated nanofiber-matrigel sandwich scaffold is designed for optimal growth and renewl of CD34+ hematopoietic progenitor cells (HPCs), which have potential applications in gene and cell therapies of the sickle cell disease. Due to the versatility of design, the scaffolds can be used to expand or manipulate other types of stem cells by incorporating the necessary growth factors, and has broader applications in disease treatment.
|Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia et al. (2016) Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides. Sci Rep 6:32681|