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First published online 1 June 2004
doi: 10.1242/jcs.01103

Journal of Cell Science 117, 2971-2981 (2004)
Published by The Company of Biologists 2004
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Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential

Gianluca D'Ippolito1, Sylma Diabira1,2,3, Guy A. Howard1,4, Philippe Menei2,3, Bernard A. Roos1,5 and Paul C. Schiller1,4,*

1 Geriatric Research, Education, and Clinical Center and Research Service, Veterans Affairs Medical Center and Department of Medicine, University of Miami School of Medicine, Miami, FL 33125, USA
2 Département de Neurochirurgie, Centre Hospitalier Universitaire d'Angers, 4 rue Larrey, 10 rue André Bocquel, 49100 Angers, France
3 INSERM ERIT U646, Ingénierie de la Vectorisation Particulaire, 10 rue André Bocquel, 49100 Angers, France
4 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33125, USA
5 Department of Neurology, University of Miami School of Medicine, Miami, FL 33125, USA



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  		Fig. 1. MIAMI cells. (A) The number of colonies obtained after density gradient centrifugation (black bars) was significantly smaller than the number of colonies obtained without centrifugation (white bars) after plating the same number of bone marrow cells per dish using the conditions for expansion/selection of MIAMI cells. This result was observed in all cultures examined independent of age. The graph shows the mean±s.e.m. of more than 15 representative experiments. (B) MIAMI cells are enriched by a unique expansion/selection procedure to isolate a population of small (7-10 µm), highly proliferative cells with reduced cytoplasm. Essential in this procedure is a unique growth environment, with sequential culturing steps under defined oxygen tension, cell density, growth factors, serum concentration and substratum conditions. (C) MIAMI cells plated at low density (0.1-0.2 cells/cm2) were allowed to proliferate (D) and when single-cell-derived colonies reached a size of 50-100 cells (E) the colonies where isolated using cloning rings, replated and expanded again at low density. The morphology of the cells is maintained after 5 cell doublings (F) and 52 cell doublings (G). (B,F,G) 20x magnification; (C-E) 10x magnification.

 


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  		Fig. 2. Characterization of MIAMI cells. (A) MIAMI cells were harvested and labeled with antibodies against CD29, CD36, CD49E, CD54, CD56, CD63, CD81, CD90, CD122, CD164, CNTFR, and HLA-DR or control IgGs as indicated and analyzed by FACS. Plots show isotype control IgG-staining profiles (black lines) versus specific antibody staining profile (colored lines). (B) Ten micrograms of total protein was analyzed by western blot using an antibody that recognizes human cMet. Expression of the 190 kDa precursor and 140 kDa cleaved receptor was detected in MIAMI cells and in the PC-3 prostate cancer cell line (positive control). (C,D) RT-PCR analysis of expression of the stem cell markers Oct-4 and Rex-1 (C) in MIAMI cells and of hTERT mRNA in two separate MIAMI cell isolates (D, RNA isolated from the human foreskin fibroblast cell line hTERT-BJ1 was used as positive control; +ve cont). PCR products of specific sizes corresponding to each gene were obtained. RNA-specific amplification is demonstrated by the absence of a band when RT was excluded (- RT).

 


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  		Fig. 3. Expansion capacity of long-term cultures of MIAMI cells. More than 50 cell doublings without detectable differentiation could be obtained in cultures grown in medium containing low serum (2%) supplemented with 15% conditioned medium (2% serum). Cells cultured for a few passages or cells expanded in excess of 50 population doublings remained small, with reduced cytoplasm, and differentiated into osteoblastic and neural phenotypes. Cells were counted at each passage with a hemacytometer.

 


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  		Fig. 4. Osteoblastic induction of MIAMI cells. (A) Alizarin Red-S staining of hydroxyapatite-associated calcium mineral deposited in the extracellular matrix by osteoblastic cells derived upon osteogenic induction (see Materials and Methods) of MIAMI cells. All plated cells induced to differentiate developed properties of functional osteoblasts. (B) RT-PCR analysis of transcripts present in osteoblast-induced MIAMI cells. MIAMI cells (lanes 1-4: different isolates) induced to differentiate to the osteoblastic lineage express the osteoblast phenotypic markers bone sialoprotein (BSP), osteocalcin (OC), runt-homology domain transcription factor Runx2, and osteopontin (OP). The size of the human-specific PCR products and the number of amplification cycles are in parentheses. BSP and OC transcripts were not detected in uninduced MIAMI cells (not shown).

 


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  		Fig. 5. Adipogenic induction of MIAMI cells. (A) Sudan-IV staining of triglyceride lipid droplets accumulated in the cytoplasm of adipocytic cells derived upon adipocytic induction (see Materials and Methods) of MIAMI cells. Functional differentiation was observed in all cells plated. (B) RT-PCR analysis of transcripts present in adipocyte-induced MIAMI cells. MIAMI cells induced to differentiate to the adipogenic lineage express the adipocytic phenotypic markers lipoprotein lipase (LPL) and pro-adipocytic transcription factor peroxisome proliferator activated receptor {gamma}-2 (PPAR-{gamma}2), detected after 25 amplification cycles. These transcripts were not detected in uninduced MIAMI cells (not shown).

 


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  		Fig. 6. Neural induction of MIAMI cells. Neural-competent cells require a sequential neuro-induction process of specification (step 1; bFGF), commitment (step 2; ßME/NT-3), and differentiation (step 3; NT-3/NGF/BDNF) (see Materials and Methods). Each stage is characterized by the stage-specific expression of specific markers. Nestin (top left) is expressed first, followed by induction of ß-III-tubulin (TuJ1) and NGF receptor (middle) in about 50% of the cells, and then expression of neurofilament-160 and NeuN (bottom) in nearly 40% of plated cells. Morphologically homogeneous cell populations are observed after each step.

 


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  		Fig. 7. MIAMI cells can be induced to develop features resembling those of pancreatic islets. MIAMI cells growing as adherent cultures can be induced to grow in an attachment-independent fashion, forming spherical or oblong clusters (A,B). Grown under endodermal-promoting conditions (see Materials and Methods), (C) MIAMI cells can be induced to express transcripts for insulin and glucagon, assayed by PCR of reverse transcribed (+RT) or control (-RT) RNA isolated from control (Cont) or treated (Tx) MIAMI cells or islet-positive (hIslet) controls. These islet-specific transcripts were not expressed under uninduced conditions (not shown). Control (Cont) and treated (Tx) MIAMI cells expressed Beta2/NeuroD (295 bp), ISL1 (542 bp), and Nkx6.1 (239 bp) transcripts (C). In all cases, bands were detected after 25 amplification cycles. MW, molecular size markers; Fk, forskolin.

 

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© The Company of Biologists Ltd 2004