Dynamic Changes within Neighborhoods of Genes Expressed by Marrow CD34+ Cells during Recovery from Radiation Injury.

The dynamic cellular and molecular responses of marrow stem / progenitor cells (SPC) during recovery from radiation injury were investigated in non-human primates. Total body irradiation (TBI) was delivered to baboons which resulted in prolonged hematopoietic suppression but ultimately endogenous recovery occurred by day 35–49. The marrow CD34+ cells obtained 28 days following TBI represent a primitive subpopulation of SPC since they were largely incapable of forming hematopoietic colonies (4.5 fold less than pre-irradiated level) or cobblestone areas yet they were responsible for hematological reconstitution. In addition, the hematological origin of the day 28 CD34+ cells was confirmed by their expression of transcripts of a family of genes characteristic of cells belonging to the hematopoietic lineages including terminal deoxynucleotidyl transferase, myeloperoxidase, and myeloid transcription factor (MAFB) and GATA-2. Marrow CD34+ cells isolated on day 7, day 28, day 35, day 49, day 85 and day 115 following TBI were profiled using oligonucleotide Affymetrix arrays and the activity center algorithm was utilized to detect functional neighborhoods of genes, which identifies sets of functionally related genes that are also transcriptionally co-regulated. The marrow CD34+ cells obtained 28 days after TBI were enriched for transcripts characteristic of quiescent SPC. We observed coordinate down-regulation of a large number of genes required for cell proliferation, including splicing factors, ribosomal subunits, translational elongation factors, and chaperones. Several sets of functionally related genes were up-regulated on day 28 CD34+ cells relative to day 0 (pre-irradiated) CD34+ cells, including: (1) secreted factors and receptors (IL-11, GM-CSF, bone morphogenetic proteins and their receptors, smoothened, ciliary neurotrophic factor, stromal derived factor-1 {SDF1}); (2) the complement system proteins (C1qRp); (3) cell adhesion molecules (CD44, ICAM, matrix metalloproteinases); (4) anti-apoptotic genes (bcl-2); (5) DNA damage and repair genes; (6) PIP2 activated K+ channels; and (7) adenylate cyclases. The day 28 CD34+ marrow cells also expressed genes that are characteristic of a broad variety of non-hematopoietic tissues such as liver (complement component 1q receptor {C1qRp}, albumin, fibrinogen), brain (GABA and glycine receptors) and marrow stroma (SDF1, fibronectin). Most of the transcripts enriched in CD34+ cells obtained at later time points (day 49) encoded for proteins (terminal deoxynucleotidyl transferase, myeloperoxidase) that play important functional roles during maturation of cells belonging to multiple hematopoietic lineages. The degree of increase of transcripts detected by the microarray analyses of day 28 CD34+ marrow cells was also validated by quantitative real time PCR. These studies provide insight into the transcriptional profile of primitive bone marrow CD34+ cells and patterns of genes involved in hematopoietic recovery. This model provided an alternative means of gaining access to radioresistant, quiescent bone marrow SPC. Functional and transcriptional analysis of CD34+ cells following irradiation provides a unique opportunity to study the molecular and cellular characteristics of radioresistant marrow SPC and the events that occur as primitive marrow SPC self-renew and differentiate.