Stem-cell techniques

Autologous hematopoietic stem cells are obtained (or harvested ) from bone marrow or peripheral blood. The technique for harvesting autologous hematopoietic cells depends on the anatomic source (i.e., bone marrow or peripheral blood). A surgical procedure is necessary for obtaining bone marrow. Multiple aspirations of marrow are obtained from the anterior and posterior iliac crests until a volume with a sufficient number of hematopoietic cells is collected (i.e., 600-1200 mL of bone marrow). The bone marrow then is processed to remove fat or marrow emboli and usually is infused intravenously into the patient like a blood transfusion. 

Recently, it has been possible to grow cells of the human immune system in special mice. These mice carry a genetic defect called severe combined immunodeficiency (SCID), which leaves them with crippled immune systems, much like those in AIDS patients. Because SCID mice lack functional cellular immunity, it is possible to implant them with human cells without tissue rejection taking place. Researchers have recently developed techniques to implant human fetal tissues containing stem cells for the blood into SCID mice. It is then possible to reconstitute these mice with functional human immune system cells, including T lymphocytes and B lymphocytes. They have also found that if these SCID mice are infected by HIV, the virus will establish infection in the human tissue and destroy the T helper lymphocytes, just as it does in humans. Thus, it may be possible to study some of the mechanisms by which HIV attacks the immune system in these mice. In addition, they may be very useful for testing potential antiviral drugs. 

In November 2003, the members of the Europe Parliament voted to approve embryonic stem cell research, using techniques similar to that adopted for cloning Dolly the sheep, although severe restrictions were put in place. For US scientists, however, the US legislation meant that they were only allowed to performed research using 12 existing sources of the embryonic stem cells and were not allowed to create any new sources. 

Fig. 7.9 Intraeoronary stem cell delivery. The technique is similar to that for coronary angioplasty, which involves over-the-wire positioning of an angioplasty balloon in a coronary artery. Coronary blood flow is transiently stopped for 2-4 min while stem cells are infused under pressure,...

Murad-Netto S, Moura R, Romeo LJ, Manoel Neto A, Duarte N, Barreto F, Jensen A, Vina RE, Vraslovik F, Oberdan A, Benetti F, Saslavsky J, Vina ME, Amino JG. Stem cell therapy with retrograde coronary perfusion in acute myocardial infarction. A new technique. Arq Bras Cardiol 2004 S3 352-254 349-351. 

FFF is still a growing area of research and there are specific fields of application pushing toward innovations both in terms of instrumentation and methodology. For example, MgFFF has been developed specifically for stem cell research, and FIFFF (either FIF FIFFF or AsFlFFF and traditional FIFFF) is driven overall by pharmaceutical-biological applications. In addition, as for others analytical techniques, the new tendency for FFF is toward miniaturization of the instruments. As examples we cite the hollow fiber channel for FIFFF [49], the microthermal unit developed by Janca [50], the microthermal-electrical unit by Gale and coworkers [51], or the SPLITT cells by Hoyos and coworkers [52]. In this regard, we should say that some important but very specialized topics were not reviewed here such as the SPLITT cells and separation channels similar to FFF that are useful for preparative aims, as they would require a detailed description which has already been reported in books and reviews [53,54]. 

Three different approaches for the cultivation of isolated hematopoietic cells have been described, the static, the stirred and the immobilized culture. Static cultivation takes place in very simple culture systems like well plates, tissue-culture flasks or gas-permeable culture bags [62, 63]. As the first two systems do not allow cell cultivation on a clinical scale, the latter is actually the most often used technique for stem cell expansion. All these systems have the advantage of being easy to handle, single-use devices, which enable an uncomplicated cell harvest. But all of them do not offer possibilities for process control or continuous feeding. This causes variations in culture conditions during fermentation (e.g., oxygen tension, pH, substrate, metabolite and cytokine concentrations). 

The most sophisticated technique for stem cell expansion is the Aastrom-Rephcell system (Aastrom Biosciences Inc., Ann Arbor, Ml, USA), which is an automated clinical system for the onsite expansion of stem cells in cancer therapy. It consists of a grooved perfusion chamber for the retention of the hematopoietic cells, with the medium flow perpendicular to the channel grooves resulting in a continuous supply of fresh nutrients while metabolites are simultaneously removed [47,71,72]. This technique has already been used in a number of clinical studies [73,74]. No incompatibihty of the expanded cells was found,but the expansion of the early progenitor cells was rather low [75]. 

In their report the researchers describe a culturing technique that can turn mouse embryonic stem cells into cell clusters that resemble pancreatic islets. The clusters inner cells produced insulin, while outer cells produced glucagon and somatostatin, two other proteins typically synthesized by pancreatic cells. Most important, the embryonic stem cell-derived pancreas cells produce insulin in response to glucose, the fundamental role of beta cells that regulate insulin secretion. The major shortcoming of the system at this time is the low levels of insulin production. Refinements in culture technique or drug manipulation may be needed to achieve therapeutic levels. 

While natural heterogeneity of cancer specimens is well documented, and the ability to isolate hundreds to thousands of cells with techniques such as LCM have allowed microarray analysis of more pure populations, the recent re-introduction of the cancer stem cell theory (24,25) has presented an extreme example of the need to isolate rare populations of cells. Cancer stem cells appear to exist at a frequency on the order of 1 in 1000 cells (26). While obtaining completely pure populations of stem cells remains difficult, highly enriched populations of these cells can be isolated using flow cytometry separating on the basis of the presence of certain specific cell markers. 

While laser capture microdissection and other related techniques may allow for the generation of relatively homogenous cell populations, most such purification steps will largely eliminate any potential cancer stem cells, because these techniques necessitate the identification of differentiated cell types for isolation. As such, cancer stem cells would frequently be eliminated from analysis, and any cancer stem cells that may be accidentally included in the analysis would be in such low occurrence that their signature would most certainly be missed. 

Each of the aforementioned amplification strategies is of potential use in studying smaller, purer cell populations however, few if any of these techniques are amenable to the study of single cells, which may be necessary if analyzing cancer stem cells. In order to study individual cells, it is necessary to have a technique that is sensitive down to the... 

Bakshi, A., Hunter, C., Swanger, S., Lepore, A., Fischer, I. (2004). Minimally invasive delivery of stem cells for spinal cord injury advantages of the lumbar puncture technique. JNeurosurg Spine, 1,330-7. 

---