Stem cells (SCs) are primitive cells with the potential to differentiate into mature cells 1. An increase in SCs is observed after various events such as myocardial infarction 2, dilated myocardiopathy 3, cardiac surgery with cardiopulmonary bypass 4, twelve weeks of physical exercise 56, menstruation 7, cessation of smoking 8, and in animals or human cells subjected to deep hypoxia conditions in vitro 9101112.

Several studies have found that elevated concentrations of SCs correlate with better clinical outcomes 13, since they possess a general regenerative capacity in blood vessel disorders 14. Various methods of SC delivery have been shown to be beneficial, mostly with autologous bone marrow cell transplantation 151617. No significant differences were found when bone marrow cells or SCs from peripheral blood were compared 18, nor when the comparison was made between bone marrow cells and adipose tissue-derived SCs 19.

An EPO-induced increase of hematopoietic stem cells (HSCs) has been detected in healthy individuals and in patients with renal anemia at two weeks post-administration 20. Moreover, an EPO-induced mobilization and homing of HSCs and their mediated neovascularization has also been reported in rats after post-myocardial infarction heart failure after six weeks of treatment 21.

Historically, intermittent hypoxia exposure sessions have been used to improve the physical condition and to treat several illnesses, mostly in the countries of the former Soviet Union, although this has been done without a clear understanding of their holistic effects 22. At all events, this practice has now become widespread in the sport world, and there are even several commercialized forms. Hypoxia exposure has been combined with normal athletic training according to different patterns 23, the most widely-adopted at present being the living-high training-low model 24.

The different forms of standard physical exercise can be difficult to apply with hypoxic procedures, especially in some patients with severe obesity, osteoarticular conditions, neurological sequelae, etc. In contrast, muscle electrostimulation can be easier to apply and has been shown to be as efficient in mimetizing training effects 252627. However, intermittent hypobaric hypoxia exposure has been demonstrated to be an efficient stimulus for eliciting adaptive responses in myocardium 28 and skeletal muscle 29.

The aim of the present study was to determine whether it was possible to increase blood SC concentration by means of: 1) short-term intermittent hypoxia, at levels well tolerated by healthy humans and previously demonstrated by our group as being capable of increasing EPO and stimulate erythropoiesis 30 and 2) muscular electrostimulation alone or combined with the aforementioned hypoxia.

Only the HME experimental data set showed a clear increase for all the subjects (about 3× fold) in the percentage of circulating CD34⁺ cells, although no significant differences were detected (p = 0.056). However, the number of circulating CD34⁺ cells increased in this experiment from a median value of 0.95 cells·μL^-1 (range: 0.5-2.1) to reach a median level of 6.65 cells·μL^-1 (range: 3.7-10.7), this increase being clearly significant (p = 0.009) (Figure 1).

No other studied parameter showed changes in this experimental block. Furthermore, neither OH nor OME experimental data showed statistically significant changes across the study for general leukocyte parameters or circulating CD34⁺ cells (Table 1).

The main result of the present study is the synergic capacity of a short-term intermittent hypoxic stimulus plus surface-electrode muscle electrostimulation to increase the circulating concentrations of hematopoietic CD34⁺ stem cells in a group of four healthy men aged around 50 years old. This increase can be considered as substantial, because it is generally accepted that a concentration of 7 cells/μL is equivalent to approximately 5·10⁵ cells·kg^-1 in an adult subject. This concentration can be assumed to be useful for harvesting purposes and corresponds to a considerable fraction of the increase in CD34⁺ cells obtained after a standard five-day treatment involving two-day doses of G-CSF (personal data).

It also seems that the increases in CD34⁺ produced by G-CSF have a non-progressive tendency, as reported in a study of patients with myocardial infarction, in whom circulating CD34⁺ levels began to decrease the day after the fourth consecutive dose of G-CSF, reaching the previous concentrations between days 6 and 10 after the end of G-CSF treatment 33. In the present study, CD34⁺ levels appear to continue increasing 7 days after the last hypoxia session, and thus it is not clear if a plateau or maximum value has been reached. It should also be taken into account that G-CSF shows some pro-thrombotic effects3435.

The lack of response in the OHE experiment does not seem attributable to the age of the study participants, since a clear HSC response to physical exercise was detected in a group of 63-year-old men 6. However, there are alternative explanations for these findings: 1) the relatively short duration of the hypoxic stimulus (a total of 9 h), whereas positive neurogenesis in rats was demonstrated after applying a hypoxic stimulus of 4 h per day over two weeks 9, while other studies detected a positive SC response to physical exercise after about three months of routine physical activity 56; at all events 7 days are enough after myocardial infarction to increase the number of CD34⁺ cells 36 and a single intense exercise test is able to increase HSC 24-48 h after an exercise bout 3738; or 2) the low intensity of the stimulus in our study (used in order to be applied and tolerable to a large majority of healthy people) compared with some in vitro studies, in which clearly more hypoxic atmospheres were used 1011. Obviously, a higher number of repeated hypoxia sessions could be applied; however, it does not seem reasonable to use much more intense (higher simulated altitude) or longer hypoxic sessions as these might not be tolerated by some people or patients.

It is also worth noting some of the advantages of muscular electrostimulation over exercise during hypoxia exposure: a) it is easy to measure and reproduce; b) it can be applied in a hypoxic atmosphere (hypobaric chamber or breathing a hypoxic mixture); and c) it can be applied to the majority of humans, even those with mild or severe physical limitations for standard exercise. It is not clear from the present study whether muscular electrostimulation should necessarily be applied simultaneously during hypoxia exposure.

The major limitations of the present study are the short total duration of the hypoxic stimulus in OHE (which was sufficient in HME) and the small sample size; however, given the results it does not seem very likely that a larger sample size would produce significant differences. The lack of a more complete hematologic study means we cannot rule out the possibility that the CD34⁺ increase is caused by a decrease in "homing" mechanisms in possible target tissues, although this does not seem a likely phenomenon in this case.

Regrettably, our protocol is unable to determine the optimal stimulation timing in order to produce a stable increase in CD34⁺ cells, although the apparent maintained effect observed (CD34⁺ increasing 7 days after the stimulus) suggests that some repeated "doses" might alone be enough.

Further studies are required to address several questions derived from the present research: a) the potential repercussions of the detected CD34⁺ increase on different pathologies, it perhaps being possible to increase HSC homing in injured tissues because after the release of HSCs from bone marrow, cells home to ischemic or damaged regions via alterations of the affected tissue 39; b) determining the most efficient protocols to induce an optimal and maintained increase in HSC; c) the possibility that the OH or OME stimulus applied via more persistent schedules might also induce a measurable increase in HSC; and d) the need for a more exhaustive study of the possible subclasses of SC released under HME conditions.

1) A simple protocol stimulating healthy humans with hypoxia plus muscle electrostimulation can quickly induce a notable increase in blood HSC.

2) The significant differences obtained in the HME experimental set over such a short period of time, coupled with the easy application of these two combined stimuli, make this method an interesting tool to increase efficiency in peripheral HSC collection.

This study has been performed without support form any public or private fund, agency or company. The authors declare that they have no competing interests.

GV: conception and design of the study, experimental subject, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; CJ: conception and design of the study, experimental subject, collection and/or assembly of data, data analysis and interpretation, manuscript writing; TP: conception and design of the study, collection and/or assembly of data, data analysis and interpretation, manuscript writing; JLV: conception and design of the study, experimental subject, collection and/or assembly of data, data analysis and interpretation, manuscript writing; AR: conception and design of the study, experimental subject, collection and/or assembly of data, data analysis and interpretation, manuscript writing; GMH: collection and/or assembly of data, data analysis and interpretation, manuscript writing; CA: collection and/or assembly of data, data analysis and interpretation, manuscript writing; RS: data analysis and interpretation, manuscript writing. All authors read and approved the final manuscript.