Identification and purification of mesodermal progenitor cells from human adult bone marrow

Abstract

Bone marrow-derived mesodermal stem cells may differentiate toward several lines and are easily cultured in vitro. Some putative progenitors of these cells have been described in both humans and mice. Here, we describe a new mesodermal progenitor population [mesodermal progenitors cells (MPCs)] able to differentiate into mesenchymal cells upon appropriate culture conditions. When cultured in presence of autologous serum, these cells are strongly adherent to plastic, resistant to trypsin detachment, and resting. Mesodermal progenitor cells may be pulsed to proliferate and differentiate by substituting autologous serum for human cord blood serum or fetal calf serum. By these methods cells proliferate and differentiate toward mesenchymal cells and thus may further differentiate into osteoblats, chondrocytes, or adipocytes. Moreover MPCs are capable to differentiate in endothelial cells (ECs) showing characteristics similar to microvessel endothelium cells. Mesodermal progenitors cells have a defined phenotype and carry embryonic markers not present in mesenchymal cells. Moreover MPCs strongly express aldehyde dehydrogenase activity, usually present in hematopoietic precursors but absent in mesenchymal cells. When these progenitors are pulsed to differentiate, they lose these markers and acquire the mesenchymal ones. Interestingly, mesenchymal cells may not be induced to back differentiate into MPCs. Our results demonstrate the adult serum role in maintaining pluripotent mesodermal precursors and allow isolation of these cells. After purification, MPCs may be pulsed to proliferate in a very large scale and then induced to differentiate, thus possibly allowing their use in regenerative medicine.

FIG. 1.

Morphological characterization of cells from autologous serum cultures. Culturing bone marrow mononuclear cells in autologous serum for ∼15 days allows the identification of two major adherent cell populations. The use of contrast–phase microscopy (A) reveals the usual MSCs fibroblastoid population and, in addition, rounded cells, strongly rifrangent in the core region, with a fried egg appearance. Scanning electron microscopy (B) reveals that these cells are thick in the core region and surrounded by a complex of microvillus structures.

FIG. 2.

MSCs MPCs-derived: proliferative and clonogenic potential. (A) Digestion of hAS culture by trypsin/EDTA allows the isolation of MPCs (trypsin resistant) from primary MSCs. If subsequently cultured in FBS, cells are able to sustain a confluence of fibroblastoid cells (T1) that show tri-lineage mesodermic differentiation potential. Once MSCs are detached from T1 culture, some MPCs remain undetached and are capable of producing a second confluence of MSCs (T2). This procedure could be repeated; however, some MPCs remain undetached. (B) Shows one of five experiments where the life-span of T1, T2, T3, T4, and T5 cultures is similar to primary MSCs cultured on FBS; however, the clonogenic potential decreases. For all experiments we were able to obtain at least a T5 with similar results.

FIG. 3.

Tri-color immunofluorescence characterization of MPCs. Staining of autologous serum cultures shows two main cell populations, easily distinguishable by shape and positive stain for CD90. Mesodermal progenitor cells are defined as rounded and negative for CD90, differing from fibroblastoids, CD90 positive MSCs. The figure shows that SSEA-4 is strongly expressed by MPCs (A) and is rarely on MSCs (yellow on merged picture indicates double positive, green and red, stain), while TRA-1–81 is exclusively expressed by MPCs (B). Furthermore, most of the growing MSCs show intense nuclear fluorescence when stained for Ki-67 (C) but none of MPCs. Panel (D) shows negative stain for CD133 on both MPCs cells and MSCs.

FIG. 4.

Characterization of TrypLE Select–detached cells from FBS or hAS cultures. Cells from FBS cultures (A) detached by trypsin alternative proteases show typical MSCs scattergram with low levels of SSC signals. Events on region R1 express bright levels of CD105 and CD90. In detached hAS cultures (B) we define the R3 population as characterized by high SSC signals. R3 gated events express a dim positive stain to CD105 and are negative to CD90. ALDH activity is present in MPCs while almost completely absent in MSCs as shown both by citofluorimetric analysis (Aldefluor) and by cytochemistry (green in the picture, where CD105 red is present in ALDH-negative cells only). Pictures of cytospins from hAS culture (C) stained by May Grünwald-Giemsa, show two population morphologies. MSCs appear with a low nucleus/cytoplasm (N/C) ratio and, often, with polylobular nuclei. It is also possible to identify cells with higher N/C ratio (arrows), intense basophilic cytoplasm, rounded nuclei, and with lots of membrane extrusions.

FIG. 5.

Detection of embryonic markers Oct-3/4 and Nanog. Mesodermal progenitors cells (MPCs) appear rounded and with not organized globular actin (red in A and C). Furthermore, cells show a bright positive stain for Oct-3/4 (A) and Nanog (C) into the nucleus. The nuclear localization of green fluorescence is confirmed by confocal microscopy in spectral mode (B and D) where nuclei of MPCs are identified by negative CD90 stain (arrows). In contrast, co-cultured MSCs show typical spindle-shaped morphology and well-organized F-actin cytoskeleton (A and C). MSCs' nuclei are negative for Oct-3/4 and Nanog. DAPI: 4′,6-diamidino-2-phenylindole. RT-PCR analysis (E) reveals, in all three assays performed, high expression of OCT3/4 on MPCs and low level on trypsin-detached cells (MSCs), while NANOG is exclusively expressed by MPCs.

FIG. 6.

Single MPC-derived fibroblastoid colony. Microscopic fluorescence picture shows bright stain for SSEA-4 (green) in the central area, while cells from periphery express high levels of CD90 (red) but not SSEA-4. Nuclei were stained with DAPI (blue).

FIG. 7.

VEGF stimulation of capillary-like network formation by mesodermal progenitor cells (MPCs). Mesodermal progenitor cells plated in six-well culture plates (4 × 10⁴ cells/well) pre-coated with Matrigel were incubated with various concentrations of VEGF (50 ng/mL yield ideal angiogenesis) for 16, 24, and 48 h. Photographs were taken at 10× magnification. Maximal response was obtained at 24 h after the addition of VEGF (A). Capillary length was measured by image analysis using Image Pro software as described. Results of capillary length after 24 h as a function of VEGF or anti-VEGF are the mean ± SE; n = 6; *P < 0.001 from control VEGF-treated cells and **P < 0.05 from VEGF versus anti-VEGF-treated MPCs (B).