Spatial gradients of blood vessels and hematopoietic stem and progenitor cells within the marrow cavities of the human skeleton

Abstract

This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34 ؉ and CD117 ؉ hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. IntroductionQuantifying the spatial profile of blood vessels and primitive cell populations most susceptible to myelosuppression or hematologic toxicity is of major interest in basic cancer research, bone marrow transplantation, and molecular radiotherapy. [1][2][3] Although several groups have investigated the spatial location of hematopoietic stem and progenitor cells (HSPCs) in murine models, 4-7 evidence for corresponding HSPC spatial profiles within human bone marrow has only recently been established. 8 In their study, Watchman et al used novel methods for the digital quantification of the CD34 ϩ hematopoietic stem cells and blood vessel fragments (BVFs) in human iliac crest. 8 The present study uses similar techniques of immunohistochemistry and digital image processing to directly measure the concentration of BVFs and CD34 ϩ HSPCs as a function of distance from the most proximal trabecular surface in human bone marrow. The study further advances the findings of Watchman et al, however, through (1) immunohistochemical stratification of the additional population of CD117 ϩ hematopoietic stem and progenitor cells, (2) consideration of a possible bone-site dependence of the BVF and HSPC spatial gradient, (3) use of larger field-of-view marrow specimens through autopsy harvest, and (4) explicit consideration of active versus total bone marrow area. MethodsPostmortem bone samples were collected during the autopsy of 9 recently deceased patients determined to be absent of marrow disease under a Health Insurance Portability and Accountability Act-compliant and University of Florida institutional review board-approved protocol that followed the Declaration of Helsinki provisions. Sections of bone were collected from the right iliac crest, L 1 vertebrae, and 1 left rib, within 24 hours of death. Excised bone samples were placed in stock formaldehyde, rough sectioned, decalcified, and acid neutralized following the manufacturer's instructions (Formical-4 and Cal-Arrest; Decal Chemical Corp). The paraffin tissue blocks were faced and 4 sequential sections were collected from each specimen at a thickness of 5 m. ImmunohistochemistrySerial sections collected from decalcified, paraffin-embedded blocks were manually immunostained using mouse anti-CD34 (dilution 1:25, QBEND10; DAKO Cytomation) and rabbit anti-CD117 (dilution 1:300, c-Kit; DAKO Cytomation). Antigen retrieval was achieved using heat-induced epitope retrieval and treatment of slides with 10 mM Citra buffer (pH 6.0), after which slides were stained by the ABC-Elite method (Vector Labs) following the manufacturer's instructions. Positive signal was detected with dia...

The relationship between bone, hemopoietic stem cells, and vasculature

“…Micro-CT permitted noninvasive imaging of the metaphyseal trabeculae in 3 dimensions and demonstrated that HSPCs in the metaphysis exist in an endosteal niche, as previously defined by us and others as within 12 cell diameters from the bone. 3,18,[22][23][24] Histomorphometric analyses of BVs in resin sections showed that all HSPCs within the metaphysis are Ͻ 3 cell diameters from a BV. Taken together, these data demonstrate that HSPCs within the metaphysis exist in an endosteal niche where they lie in close proximity to BVs.…”

“…In our and other laboratories, [22][23][24] the endosteal region is arbitrarily defined as within 12 cell diameters (ϳ 96 m) of bone. 3 Classifying transplanted HSPCs within trabecular-rich regions of bone as either central or endosteal cannot take into account trabecular bone lying just outside the plane of section.…”

Three dimensionalde novomicro bone marrow and its versatile application in drug screening and regenerative medicine

“…82 Although the 3D architecture of the BM tissue in situ remains largely uncharacterized, [83][84][85][86] quantitative analysis of the interactions between these independent and dependent variables using computational modeling can aid in determining which components are more important for the specific functions of the in vivo B/BM system under normal or pathological conditions. [87][88][89] This in-depth understanding can guide the design of 3D de novo micro BM and advance investigations into disease processes using the 3D BM disease models.…”

Skeletogenesis and the Hematopoietic Niche

“…In contrast, there is also evidence that the osteoblast and vascular niches provide different roles in hematopoiesis, e.g. a quiescence and maintenance role verses a differentiation and egress role (Lord et al 1975;Shackney et al 1975;Gong 1978;Nilsson et al 2001;Heissig et al 2002;Arai, F. et al 2004;Balduino et al 2005;Jang and Sharkis 2007;Bourke et al 2009). Further, although these niches are proximal in the chondro-oseous region, mathematical modeling has predicted a layer of only two myeloid cells is sufficient to deplete most oxygen provided by a near by sinusoid (Chow et al 2001).…”

“…However, at least two hematopoietic niches have been described, an osteoblastic (or endosteal) niche, ascribed to osteoblasts residing on bone surfaces, and a vascular niche, ascribed to endothelial cells and subendothelial MSCs or pericytes lining marrow sinusoids. Many have argued that more quiescent LT-HSCs and ST-HSCs are located in the osteoblast niche, while differentiating HPCs are located in the vascular niche for mobilization to the periphery (Lord et al 1975;Shackney et al 1975;Gong 1978;Nilsson et al 2001;Heissig et al 2002;Arai, F. et al 2004;Balduino et al 2005;Jang and Sharkis 2007;Bourke et al 2009). However, these regions in the marrow are so close in proximity (Arai, F. et al 2004;Kiel et al 2005;Lo Celso et al 2009;Xie et al 2009), that the osteoblast and vascular niches may be the same, or perhaps interchangeable to some degree.…”