Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells
Christina Holzwarth
0
3
Martin Vaegler
0
3
Friederike Gieseke
0
3
Stefan M Pfister
2
Rupert Handgretinger
0
3
Gunter Kerst
1
Ingo Mller
0
3
0
University Children's Hospital, Department of General Pediatrics, Hematology and Oncology
,
Tubingen
,
Germany
1
University Children's Hospital, Department of Pediatric Cardiology, Pulmology and Intensive Care
,
Tubingen
,
Germany
2
German Cancer Research Center and University Children's Hospital
,
Heidelberg
,
Germany
3
University Children's Hospital, Department of General Pediatrics, Hematology and Oncology
,
Tubingen
,
Germany
Background: Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results: After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1a expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion: Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.
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Background
Human multipotent mesenchymal stromal cells (MSC)
obtained from bone marrow are characterized by a
multilineage differentiation potential and a high proliferative
capacity without losing their genetic stability [1]. By the
mid of the last decade, the clinical potential was
recognized [2]. Today, several clinical trials showed that the
application of ex vivo expanded MSC is safe and feasible
[3-5]. MSC have earned considerable attention as
therapeutic tools in graft-versus-host disease after allogeneic
hematopoietic stem cell transplantation [6]. The
treatment of pediatric patients suffering from osteogenesis
imperfecta was the first successful clinical application of
MSC in regenerative medicine [3,7]. Another promising
application of MSC has been explored in a pilot study
where five children with steroid-induced osteonecrosis
of the femur received MSC directly injected into the
necrotic area of the bone [8]. Regenerative properties of
MSC could also be demonstrated in the treatment of
ischemic cardiovascular diseases [9].
These examples of tissue regeneration share the
phenomenon of oxygen deprivation in the affected organs
challenging the ability of MSC to differentiate into bone
or other tissues [10]. Physiologically the cells are
adapted to low oxygen levels with oxygen
concentrations between 1% and 7%. Mathematical models of the
pO2 distribution in human bone marrow suggest a
gradient across the marrow from the relatively well
oxygenated sinuses to the rather hypoxic endosteal region
[11]. It is known that low oxygen tension is involved in
keeping stem cells in a quiescent state retaining their
plasticity [12]. Conversely, hypoxia may also serve as a
danger signal and recruit MSC. In a rat model,
Rochefort and colleagues showed that specifically MSC and
not hematopoietic progenitor cells were mobilized out
of the bone marrow into the peripheral blood by
hypoxia [13]. However, it is an open issue if various
oxygen concentrations over prolonged periods of time
change the characteristic properties which define MSC
in vitro as proposed by the International Society for
Cellular Therapy [14]. Reports addressing this issue
obtained conflicting results: In several in vitro studies,
low oxygen concentrations have been found to be
stimulating differentiation processes, exemplarily shown by
inducing the cells toward the adipogenic, osteogenic or
chondrogenic lineage [15-17]. In contrast, other groups
reported suppressive effects of reduced oxygen tensions
on MSC plasticity [10,18]. It is hypothesized that
survival and proliferative capacity of MSC can be enhanced
by maintaining the cells under low oxygen tensions.
Standard culture of MSC at 21% O2 imply hyperoxic
conditions compared to physiological O2 concentrations,
i. (...truncated)