Identification of hematopoietic-specific regulatory elements from the CD45 gene and use for lentiviral tracking of transplanted cells

Abstract

The development of a hematopoietic reporter is crucial for determining the fate of lineages derived from cell-based therapies. A marking system will enable safer embryonic stem (ES) and induced pluripotent stem (iPS) cell-based derivation of blood lineages, and facilitate the development of efficient cellular reprogramming strategies based on direct fibroblast conversion. Here we report that the protein tyrosine phosphatase CD45 is an ideal candidate gene on which to base a hematopoietic reporter. CD45 regulatory elements were discovered by analyzing transcription factor chromatin occupancy (ChIP-seq) and promoter nuclease sensitivity (DNase-seq) to identify minimally sufficient sequences required for expression. After cloning the CD45 regulatory elements into an attenuated lentiviral backbone, we found that two transcriptional initiation regions were essential for high-level expression. Expressing CD45 promoters containing these regions and tethered to GFP in a primary B cell differentiation assay and a transplantation model resulted in high levels of GFP in lymphoid, myeloid and nucleated erythroid cells in mouse and human blood cell lineages. Moreover, high GFP levels remained five months following secondary transplantation, indicating persistence of the reporter. No CD45 driven GFP expression is observed following fibroblast or ES cell transduction. The GFP reporter is seen only after ES cells differentiate into hematopoietic cell progenitors and lineages, suggesting that this hematopoietic reporter system could be useful in validating potential autologous blood cell therapies.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene

“…There have been many efforts to generate transgenic mice with transgene expression exclusively in the hematopoietic compartment 1 . The vav1 gene has been the focus of many such studies as it is highly expressed throughout hematopoietic development from the embryonic day 11.5 (e11.5) embryo through adulthood 2 There appears to be very limited expression in other tissues in the adult mouse, with the exception of the developing tooth bud 2 .…”

Cell Therapy of Severe Ischemia in People with Diabetic Foot Ulcers—Do We Have Enough Evidence?

“…The marker CD117, also referred to as c-kit, is expressed in, for example, melanocytes, germ cells, Cajal cells, or epithelial cells [ 91 – 93 ]. Furthermore, MACs are CD45 positive but negative for the markers CD34 and CD146 [ 94 , 95 ]. These undifferentiated cells play a role in the healing of many tissues due to their ability to differentiate into different cell types [ 96 ].…”

Molecular and cellular basis of hematopoietic stem cells maintenance and differentiation

“…This would minimize the possibility of teratoma formation upon transplantation the iPSCs derived HSPCs into the host. A reliable hematopoietic reporter would only mark HSPCs but not other cell types; out of all of the candidates we tested, only CD45 met this criteria [190]. As described in Chapter II, CD45 is expressed exclusively in hematopoetic cells, and a small genomic region near the promoter was sufficent to drive high GFP expression [133-137, 145, 190, 261].…”

“…Concentrated lentiviral supernatant was produced and titered as described previously [190]. In brief, 293T cells were split for 3 consecutive days and used to package recombinant sequence into lentiviral particles using the protocol described previously [190].…”