1. by Istvàn Blazsek, Jalila Chagraoui, and Bruno Péault
Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow
by Istvàn Blazsek, Jalila Chagraoui, and Bruno Péault
Blood
Volume 96(12):
December 1, 2000
©2000 by American Society of Hematology

2. Histology of the developing mouse femur
Histology of the developing mouse femur.(A) E17 femur embedded in Unicryl and stained with hematoxylin/fast green shows spongy bone.
Histology of the developing mouse femur.(A) E17 femur embedded in Unicryl and stained with hematoxylin/fast green shows spongy bone. Osteoblasts are aggregated in nodules and depose a calcified bone matrix in their surroundings. Arrows point to hematopoietic cells. (B) An abundant vascular conduit that follows the way of hypertrophied chondrocytes characterizes the newborn femur. (C) Proximal region of the femur from a young adult mouse containing spongy bone filled with bone marrow. Megakaryocytes form islands with erythroid and myeloid cells.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

3. Expansion of marrow cells and emergence of the hematon during perinatal ages.The femoral inner mass was recovered from 8 to 12 embryos or little mice.
Expansion of marrow cells and emergence of the hematon during perinatal ages.The femoral inner mass was recovered from 8 to 12 embryos or little mice. Half of each tissue was used to prepare a single-cell suspension for quantitative colony assays and the other half to count hematons and native cobblestone colonies at day 7 in LTC. The numbers of TNC (○), total GM-CFC and HPP-CFC (●), and day 7 CAFC (⋄) increased logarithmically from E17. Day 35 LTC-IC/CAFCs (♦) appear at day 2, and hematon units containing native CA (■) emerge from days 5 to 7.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

4. Adherent cells in LTC from newborn femur (A-D) and features of hematon preparations from young adult mice (E-H).Cultured marrow from newborn femur contains several adherent cell types but lacks hematopoietic foci containing CA. (A) Osteocalcine-positive ost...
Adherent cells in LTC from newborn femur (A-D) and features of hematon preparations from young adult mice (E-H).Cultured marrow from newborn femur contains several adherent cell types but lacks hematopoietic foci containing CA. (A) Osteocalcine-positive osteoblasts surrounded by hematopoietic cells. (B) A tartrate-resistant acid phosphatase–positive giant, multinuclear osteoclast surrounded by macrophages. (C) Cell island containing MMP-9/gelatinase-B–positive cells. (D) Firmly adherent αSM-actin–positive stroma cells at day 14 in culture. In young adult marrow, hematons resemble a bunch of grapes around capillaries after removal of the bulk buffy coat suspension. (E) Stereomicroscope view. (F) Phase-contrast microscope view. (G) Purified fraction of the hematon core complex. (H) More than 90% of core complexes develop in native CA at days 2 to 7 in culture.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

5. Developmental age-related gain of autonomous cell production ability in the femur.Total cells from femora were dissociated and seeded in triplicate LTCs, and nucleated cells, GM-CFU, and HPP-CFC were measured over time.
Developmental age-related gain of autonomous cell production ability in the femur.Total cells from femora were dissociated and seeded in triplicate LTCs, and nucleated cells, GM-CFU, and HPP-CFC were measured over time. All 3 cell populations declined rapidly between E17 through day 5. Productive cultures were obtained only from mice aged older than 7 days. E18 (▿), NB (■), postnatal (PN) day 5 (○), day 7 (◊), day 14 (■).
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

6. Age-related gain of stromal cell competence
Age-related gain of stromal cell competence.Stromal layers were prepared from the femur at different ages, then their capacity to support day 7 CAFC and day 35 LTC-IC/CAFC from normal marrow was compared using LDA. Single-cell suspensions of reporter cells ...
Age-related gain of stromal cell competence.Stromal layers were prepared from the femur at different ages, then their capacity to support day 7 CAFC and day 35 LTC-IC/CAFC from normal marrow was compared using LDA. Single-cell suspensions of reporter cells were seeded in 24 replicates at each of the 4 dilutions. Log percentage of wells negative for CAFC are plotted against initial cell input and 1× values were intrapolated at the −37% value. (A) Day 7 CAFC. (B) Day 35 LTC-IC/CAFC. Symbols represent stromal cells prepared at different postnatal ages: day 5 (○), day 7 (■), day 8 (⋄), day 10 (●), 2 months (⧫). The MS-5 cell line (*) was used as a control reference.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

7. Myelopoietic and B-lymphopoietic capacity of buffy coat and hematon in LTC.TNC and CFU were determined from the nonadherent (NA) and adherent cell fractions (AD).
Myelopoietic and B-lymphopoietic capacity of buffy coat and hematon in LTC.TNC and CFU were determined from the nonadherent (NA) and adherent cell fractions (AD). Buffy coat (A) and hematon (B) cultures. Commutation from myelopoiesis (Dexter conditions) to B lymphopoiesis (Whitlock-Witte conditions) in LTC was measured by analyzing the GM-CFU and B220+ cell populations. TNC-NA (○), TNC-AD (●), GM-CFU-NA (■), GM-CFU-AD (▪), HPP-CFC-NA (▵), HPP-CFC-AD (▴), B220+ B lymphocytes (*). Representative experiments repeated 3 times with similar results are shown. Data points represent the mean (±1 SEM) of 4 determinations.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology

8. Stromal and hematopoietic cell composition of the hematon core complex
Stromal and hematopoietic cell composition of the hematon core complex.The hematon core complex fraction was purified from young adult mice, washed in PBS/0.5% BSA/0.1% Na azide, and cytocentrifuged.
Stromal and hematopoietic cell composition of the hematon core complex.The hematon core complex fraction was purified from young adult mice, washed in PBS/0.5% BSA/0.1% Na azide, and cytocentrifuged. Immunostaining of native preparations with antibodies to Stro-1, desmin, Flk-1, Sca-1, VLA-4, CD34, Thy-1, c-kit, CD41, and acetylcholinesterase reaction on adherent core complexes at day 4 in culture show the discrete localization of cells within the hematon core complex or in close contact with the vascular endothelium.
Istvàn Blazsek et al. Blood 2000;96:
©2000 by American Society of Hematology