Adult Human Stem Cells in Regenerative Medicine

By: Dr. Gerhard Bauer, BioFiles 2008, 3.11, 6.

BioFiles 2008, 3.11, 6.

Dr. Gerhard Bauer
Laboratory Director GMP Facility
Adjunct Assistant Professor Stem Cell Program
School of Medicine, University of California Davis

Stem cell therapies offer hope to thousands of people suffering from diseases or injuries that destroy or damage vital cells and tissue. Loss of such tissue is invariably linked to loss of function, which often translates into severe disability. In spite of this hope, just the debate over stem cells has also stirred quite some controversy, particularly in the general public. In this article we will give a definition of stem cells, describe functional properties of adult stem cells, and outline some of their immediate clinical applications in the field of regenerative medicine.

In general, stem cells can be described as a reservoir of nondifferentiated cells that can give rise to all kinds of differentiated (mature) cells with specific functions. Three main features define this cell population: Self Renewal, Proliferation and Multi-Potentiality.

To illustrate the necessity for such cells in long lived organisms, the hematopoietic or blood forming system in the human body is a good example. Mature blood cells are highly specialized cells and only live for a short period of time. A red blood cell, for instance, has an approximate life span of 120 days, after which it will be eliminated. Billions of red blood cells circulate in our vascular system; an inexhaustible source of blood cells is needed, with its root being the hematopoietic stem cell. It has all the properties described earlier. It makes more of itself, while it also allows for the production of differentiated “progenitor cells”, which will differentiate into mature blood cells of all lineages (red and white). Hematopoietic stem cells fall into the category of “adult stem cells”.

Two groups of stem cells can be described: Adult type stem cells, which are found in fetus, infant, and adult, and embryonic stem cells, only found in the first few days of the development of an embryo. While adult stem cells are designated stem cells producing cells of only one particular tissue (eg.: skin, liver, blood), embryonic stem cells hold the distinct advantage of being able to produce all cells and tissues of the body, including nerve and brain cells.

Adult, hematopoietic stem cells are found in the bone marrow, but also in the umbilical cord blood of a newborn infant. Blood formation in a developing fetus occurs mainly in the fetal liver, and hematopoietic stem cells freely circulate in the fetal blood. Only a few days after birth, the hematopoietic stem cells settle in their “adult” niche, the bone marrow space. Although hematopoietic stem cells have permanently settled, they can still be “mobilized“ from their niche by application of leukocyte stimulating growth factors (cytokines). Granulocyte Colony Stimulating Factor (GCSF) is the most widely used pharmaceutical for “stem cell mobilization”. Hematopoietic stem cells leave their bone marrow niche for a brief period of time and can be collected in the peripheral blood via a specific white blood cell collection called apheresis. This method of stem cell collection has in many centers replaced surgical bone marrow harvest, since it is less invasive for the donor and offers the collection of similar quality bone marrow stem cells. The current, most important clinical use of adult, hematopoietic stem cells is allogeneic bone marrow transplantation for the treatment of leukemias. To cure leukemias, chemotherapy and radiation will be given to a patient, which dramatically reduces the number of leukemic cells.

However, complete elimination of all leukemic cells is an absolute must to prevent relapse of the cancer. To achieve this goal, an immunological component is added. With the transplanted bone marrow from another person, a completely new immune system is also transplanted into the recipient. While the patient’s cancer could evade the patient’s immune system, the newly introduced stem cells allow for a new immune system, actually the donor’s immune system, to grow back, which now recognizes leukemic cells as foreign and eliminates them. Properly executed, an allogeneic, hematopoietic stem cell transplantation procedure promises an 80% cure rate of certain leukemias.

Many other adult type stem cells can be found in the human body. Skin and gut cells need to renew continuously, therefore stem cells are needed for this purpose. Hematopoietic stem cell cells, in their niche, are attached to “stroma” cells (support tissue), which only recently were identified as another subset of human stem cells now called “mesenchymal stem cells” (MSC). Besides in bone marrow, MSC can be found in adipose tissue, and also in the placenta and the vicinity of the blood vessels in the umbilical cord. MSC can differentiate into bone, cartilage and muscle. It has therefore been suggested that such cells could be used for tissue repair purposes.

Also Endothelial Progenitor Cells (EPCs), stem cells that line blood vessels and are thought to be important for vessel formation, were suggested to be useful clinically for regeneration of vasculature. However, EPCs are difficult to isolate and to culture, and not many applications of EPCs have been followed through clinically.

At the same time, hematopoietic stem cells (HSC) were found to have a different, but very interesting other property: Besides reconstituting the blood forming system, they were found to also help with regeneration of other tissues. If injected into an animal with a tissue injury, isolated hematopoietic stem cells migrate to the site of injury and induce tissue regeneration, most likely by new blood vessel formation and endogenous tissue stem cell recruitment by trophic factors. So which cells are the most appropriate stem cells to be used in regenerative medicine? MSC, EPC, HSC or a mixture? Can all of these cells participate in tissue repair? Will they become damaged tissue, and what are their homing properties?

To follow up on these properties in vivo, we developed a novel mouse model, the NOD SCID MPSVII mouse, which allows the tracking of injected human cells by a simple chemical stain. The deficiency in β-glucuronidase (GUSB) results in a lysosomal storage disease called Mucopolysaccharidosis Type VII. Successive crossing of MPSVII mice onto the NOD/SCID background has produced an immune-deficient mouse that facilitates tracking of GUSB containing stem cells, either human or wild type mouse (Hofling et al, Blood 2003). Injected stem cells are visualized by a bright red enzymatic stain on tissue slides; tissues can be counterstained to visualize nuclei of the endogenous mouse tissue. The GUSBdeficient NOD/SCID/MPSVII mouse allows the sensitive detection of individual unmarked cells in engrafted tissues by normal levels of the enzyme, without reliance on the continued expression of human cell surface markers or in situ hybridization.

Data from our lab show that injected adipose and bone marrow derived MSC lodge in multiple tissues following various routes of administration into sublethally irradiated immune deficient mice. Yet in models of acute local injury, MSC appear to preferentially home to, or accumulate in the damaged tissue. Not only MSC follow this pattern, also HSC, injected intravenously, accumulate at the site of injury.

What attracts a Mesenchymal Stem Cell into a damaged region of tissue? We are examining hypoxia, Stromal Derived Factor -1 (SDF-1), and Hepatocyte Growth Factor (HGF), as major contributing factors; also hypoxia in the area of injury seems to play a major role.

For isolation of adult stem cells, what markers could be used?

Surface Markers

Endothelial Progenitors (EPC)

CD31+/CD34+/CD45-, but also have different phenotypes

Hematopoietic stem cells (HSC)

CD45+

In general, thought to be CD34+/CD38-, but CD34 is reversible,also CD133+

Mesenchymal stem cells (MSC)

CD45-
CD105+: also on EPC and HSC
NGFR+ and -
CD133+ and -
CD133+ and -

These and other markers are externalized and internalized, regulated by hypoxia, pH, Nitric Oxide, ligands, and other micro environmental factors.

We also sought a marker of conserved stem/progenitor cell function that transcended cell surface phenotype, and did not restrict cells to a “cell type”, such as HSC vs. MSC. The enzyme Aldehyde Dehydrogenase (ALDH) was a safe candidate, which had non-toxic isolation methods available. ALDH hi cells have robust hematopoietic capacity in vivo, and also contain endothelial and MSC early progenitors.

In animal models of hind limb ischemia and coronary artery disease, the intravenous injection of purified hematopoietic stem cells lead to remarkable improvement of blood flow and function. Results in such models obtained in our laboratory proved to be highly statistically relevant compared to mock treated controls and demonstrated the best evidence, so far, that adult stem cell applications can be very useful in vivo. For a human clinical trial, safety of the application is of utmost importance. Therefore, excellent pre-clinical studies in animals form the basis for any human application.

In current human clinical trials, adult human stem cells are used for the treatment of peripheral artery disease (PAD), and coronary artery disease. Many more human clinical applications of adult stem cells are being evaluated currently.


Summary


Adult human stem cells used for homing into areas of tissue damage and inflammation initiate cascades of endogenous repair including re-vascularization, reduce an immune attack on damaged tissue, and tip the balance toward repair rather than scarring and fibrosis. Adult stem cells are the “paramedics of the body”—they migrate rapidly to regions of hypoxic tissue damage and inflammation, and secrete factors that enhance repair and revascularization. We do not have good evidence that they “become the tissue” that was damaged. We look to human embryonic stem cells for future direct tissue repairs and replacements. Adult stem cells, however, are very useful for increasing blood flow into infarcted hearts or poorly vascularized limbs.

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References

  1. Meyerrose, Roberts, Ohlemiller, Vogler, Nolta, Sands. Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease. MS submitted, (2008).
  2. Meyerrose, DeUgarte, Hofling, Herrbrich, Sands, Hedrick, and Nolta. In vivo Distribution of Adipose-Derived Mesenchymal Stem Cells in Novel Xenotransplant Models. Stem Cells, 25, 220-227 (2007).
  3. Levac, Robson, Neelamkavil, Hohm, Capoccia, Link, Nolta, and Hess. Aldehyde Dehydrogenase-Activity Purifies Multiple Hemangiogenic Lineages that Accelerate Vascularization of Ischemic Tissue Through Paracrine Support of Neovessel Formation. ASH 2007 abstract, Manuscript in preparation.
  4. Hess, Wirthlin, Craft, Herrbrich, Hohm, Lahey, Eades, Creer, Nolta. Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term repopulating human hematopoietic stem cells. Blood, 107(5), 2163-2169 (2005).
  5. Hess, Meyerrose, Wirthlin, Craft, Herrbrich, Creer, Nolta. Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity. Blood, 104(6), 1648-1655 (2004).
  6. Hofling, Vogler, Creer, Sands. Engraftment of human CD34+ cells leads to widespread distribution of donor-derived cells and correction of tissue pathology in a novel murine xenotransplantation model of lysosomal storage disease. Blood, 101(5), 2054-63 (2003).
  7. Wu, Nolta, Starnes, Cramer. Migration of mesenchymal stem cells to heart allografts during chronic rejection. Transplantation Mar 15, 75(5), 679-685 (2003).

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