BMJ 1999;319:1308 [full] ( 13 November )

Where are we going?

Stem cell technology

Paulo A Fontes, assistant professor of surgery.,  Angus W Thomson, professor of surgery and molecular genetics and biochemistry. 

Thomas E Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh Medical Center, 4C Falk Clinic, Pittsburgh, PA 15213, USA

Correspondence to: P Fontes fontesp{at}msx.upmc.edu

Stem cell technology is a rapidly developing field that combines the efforts of cell biologists, geneticists, and clinicians and offers hope of effective treatment for a variety of malignant and non-malignant diseases. Stem cells are defined as totipotent progenitor cells capable of self renewal and multilineage differentiation.1 Stem cells survive well and show stable division in culture, making them ideal targets for in vitro manipulation. Although early research has focused on haematopoietic stem cells, stem cells have also been recognised in other sites. Research into solid tissue stem cells has not made the same progress as that on haematopoietic stem cells. This is due to the difficulty of reproducing the necessary and precise three dimensional arrangements and tight cell-cell and cell-extracellular matrix interactions that exist in solid organs. However, the ability of tissue stem cells to integrate into the tissue cytoarchitecture under the control of the host microenvironment and developmental cues, makes them ideal for cell replacement therapy. In this overview, we briefly discuss the current research and the clinical status of treatments based on haematopoietic and tissue stem cells.


Summary points

Stem cells are progenitor cells that are capable of self renewal and differentiation into many different cell lineages
Stem cells have potential for treatment of many malignant and non-malignant diseases
Peripheral blood stem cells are used routinely in autologous and allogeneic bone marrow transplantation
Gene transfer into haematopoetic stem cells may allow treatment of genetic or acquired diseases
Embryonic stem cells may eventually be grown in vitro to produce complex organs
Neuronal stem cells are being used for neurone replacement in neurovegetative disorders such as Parkinson's and Huntingdon's diseases



    Haematopoietic stem cells
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Haematopoietic stem cells
Non-haematopoietic stem cells
Conclusions
References

Applications of cultured haematopoietic stem cells
Haematopoietic stem cells are a somatic cell population with highly specific homing properties and are capable of self renewal and differentiation into multiple cell lineages.2 Human haematopoietic progenitor cells, like stromal cell precursors in bone marrow, express the CD34 antigen, a transmembrane cell surface glycoprotein identified by the My10 monoclonal antibody.3 However, pluripotent stem cells constitute only a small fraction of the whole CD34+ population, which is by itself rather heterogeneous regarding phenotype and function. The best way to define haematopoietic stem cells is from their functional biology. They are known to restore multilineage, long term haematopoietic cell differentiation, and maturation in lethally cytoablated hosts.4 Haematopoietic stem cells can be obtained from bone marrow, peripheral blood,5 umbilical cord blood,6 and fetal liver.7

The use of peripheral blood stem cells in both autologous and allogeneic transplantation has become routine as they can be collected on an outpatient basis and also promote a consistent acceleration in haematopoietic reconstitution after engraftment.8 Umbilical cord blood stem cells have been used progressively in paediatric patients, from both related and unrelated HLA-matched donors. In recipients with severe T cell immunodeficiency disorders, fast engraftment is required together with a low risk of graft versus host disease and a low viral transmission rate.9 Since umbilical cord blood stem cells can be expanded in vitro or frozen for storage in cell banks10 they have been used in clinical trials for both autologous and allogeneic haematopoietic stem cell transplantation.11

The bone marrow is a mesenchyme derived tissue consisting of a complex haematopoietic cellular component supported by a microenvironment composed of stromal cells embedded in a complex extracellular matrix.12 This extracellular matrix has an important role in the facilitation of cell-to-cell interaction, in addition to a more complex role in the binding and presentation of cytokines to the haematopoietic progenitor cells.13 The cytokine milieu and extracellular matrix interaction provides the "road map" for maturation and differentiation of stem cells,14 which should be instrumental for their in vitro manipulation before therapeutic use. For example, haematopoietic stem cells can be manipulated in vitro to generate dendritic cells, the most potent antigen presenting cells.

Dendritic cells have a pivotal role in the elicitation and regulation of antigen specific, major histocompatibility complex-restricted T cell responses and are thought to be the only antigen presenting cells able to prime naive T cells. Dendritic cells can be derived from CD34+ precursors in response to granulocyte macrophage colony stimulating factor and tumour necrosis factor alpha  and from monocytes cultured with granulocyte macrophage colony stimulating factor and interleukin-4.15 In vitro generated dendritic cells (fig) that have been transduced with genes coding for tumour specific antigens or pulsed with tumour specific antigen or peptide could be useful for induction of cytotoxic T cell responses.16 Dendritic cell tumour vaccines could be important future therapeutic tools; phase II clinical trials are under way and show limited efficacy.17 On the other hand, the migration and function of dendritic cells derived from liver in an allogeneic environment may be seminal in the development of donor specific tolerance.18-20 Genetic engineering of dendritic cells to express immunosuppressive or immunoregulatory molecules may provide a novel method to promote graft tolerance, reducing dependence on systemic immunosuppression.21



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  		Characteristic dendritic cells derived from CD34+ haematopoietic stem cells and propagated in granulocyte macrophage colony stimulating factor

Haematopoietic stem cells and gene therapy
Haematopoietic stem cells themselves are also a promising target for gene therapy. Haematopoietic stem cells have been made resistant to one or more cytotoxic drugs22 with retroviral transfection of the multidrug resistance gene (MDR1). This should help circumvent the myelosuppressive effects of standard regimens of chemotherapy.23 Haematopoietic stem cells can also be genetically marked to allow assessment of patterns of cell survival, localisation, and function after bone marrow transplantation.24 This strategy has already been used with retroviral vectors.25 Double genetic marking is also being used to determine the long term effects of different protocols of cytokines given to promote bone marrow regeneration after cytoablative treatment.26 Gene transfer into haematopoietic stem cells represents a novel approach for treating some genetic or acquired diseases. So far, transduction of genes into humans using haematopoietic stem cells has shown low efficiency, especially in the quiescent stem cell population.27

Recent advances in reproductive biology and gene therapy have used ex vivo transduced autologous umbilical cord blood cells or direct targeting in utero as a potential means to correct haematopoietic, immunological, and metabolic single gene disorders.28 This technique has the advantage of using normal haematological development, which induces the fetus to allow space for a new cell population and promotes tolerance in the developing immune system. Unfortunately, infection and graft versus host disease are still potential risks for both the mother and the fetus. However, advances in the understanding of dose requirements and manipulation of peripheral blood sources to enrich for stem cells may provide strategies to overcome these problems.

    Non-haematopoietic stem cells
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Haematopoietic stem cells
Non-haematopoietic stem cells
Conclusions
References

The adult bone marrow also contains mesenchymal stem cells29 which are involved in the regeneration of mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, adipose tissue, and stroma. Although human mesenchymal stem cells have been isolated, it remains unclear how basal nutrients, cell density, spatial organisation, mechanical forces, growth factors, and cytokines control their differentiation.30 Isolated human mesenchymal stem cells constitute a single, phenotypically distinct population and are uniformly positive for SH2, SH3, CD44, CD71, CD90, CD106, CD120a, and CD124. They are also negative for CD14 and CD34 and can be induced to differentiate into adipocytes, chondrocytes, tenocytes, and osteocytes. Transplantation of mesenchymal stem cells into tendon defects in rabbits can significantly improve biomechanical properties of the damaged area.31

Embryonic stem cells were first isolated in 1981 through studies focusing primarily on murine blastocysts.32 Embryonic stem cells are derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro.33 Human embryonic stem cells can express high levels of telomerase activity, comparable with that expressed by cells isolated from germ lines and embryonic tissues.34 They can also form several cell types and simple tissue. Further understanding of cell tissue interactions and their relation with the extracellular matrix may eventually enable in vitro production of complex organs.35 In vitro manipulation of embryonic stem cells can be enhanced by nuclear transfer and cloning.36

Advances in stem cell technology have stimulated rapid growth in the understanding of embryonic and postnatal neural development. A population of neuronal stem cells capable of extended self renewal in addition to subsequent differentiation into both neurones and glia has already been identified.37 These common neurohaematopoietic stem cells can be isolated from the subventricular zone in the wall of the lateral ventricle of the brain. They divide in response to epidermal growth factor and fibroblast growth factor-2.38 Transplanted neuronal stem cells can integrate into an intact brain and differentiate into neuronal and glial cells. They may also function as haematopoietic stem cells when infused into irradiated mice.39 This last finding is controversial and may represent an artefact of the experimental technique, since only one contaminating haematopoietic stem cell would be needed to repopulate a mouse haematopoietic system. However, the reverse has also been shown, with stem cells derived from bone marrow entering through the brain-blood barrier, becoming fully differentiated, and displaying macrophage-like function (microglia).40

Initial clinical trials have shown that neurone replacement for neurovegetative diseases such as Parkinson's and Huntington's diseases is now feasible. Reports of transplantation of fetal striatal tissue in patients with mild to severe Huntington's disease suggest that transplantation of neuronal stem cells may improve some of the cognitive symptoms associated with the condition, as well as potentially modifying its clinical course.41 Neuronal stem cells can also be manipulated before grafting; they have been shown to respond to stimulation with fibroblast growth factor-2, and immortalised neural stem-like cells infected with viral vectors have been found to express factors that are related to neural repair.42

The existence of prostate stem cells is still a matter of debate.43 Nevertheless, the concept is interesting, mainly because of the possible analogy between effects of androgens on the prostate stem cells and that of cytokines on haematopoietic stem cells. Prostate stem cells could have important implications in the development of prostatic carcinoma.44

Understanding of liver regeneration has improved greatly since the initial description of oval cells as progeny of facultative stem cells.45 Hepatic stem cells, which are found in the interlobular bile ducts and possibly also in the canals of Hering, could have a large impact on the pathophysiology of hepatocellular carcinomas and cholangiocarcinomas. 46 47

Limbal stem cells have been described at the junction between the cornea and sclerae.48 These cells are known to be progenitors of corneal epithelium. Keratolimbal allografts are a promising treatment for bilateral blindness,49 and limbal stem cells can now be safely obtained from living related donors.50

    Conclusions
Top
Haematopoietic stem cells
Non-haematopoietic stem cells
Conclusions
References

Stem cell technology is progressing as the result of multidisciplinary effort, with clinical applications of manipulated stem cells combining developments in transplantation and gene therapy. There are rather complex ethical issues related to the applications of cloning and nuclear transfer in human stem cells. Successful ex vivo manipulation of stem cells will depend on improved understanding of the interactions between cytokines and the extracellular matrix. Cytokines may decrease binding forces between stem cells and components of the stromal microenvironment, thus facilitating the migration of stem cells into the peripheral blood. Improvements in mobilisation schedules using growth factors, stem cell isolation, and purification procedures and techniques for both positive and negative purging (to reduce tumour cell contamination or to deplete T lymphocytes) are emerging. The possibility of ex vivo multiplication of stem cells to accelerate haematopoietic recovery or to provide sufficient stem cells from one extraction to support several cycles of high dose chemotherapy is under investigation. The applications for autologous stem cell transplantation should increase as it avoids the use of non-specific immunosuppressive therapy. Peripheral blood stem cells have advantages over bone marrow cells for autologous transplantation in that they show consistently faster haematopoietic reconstitution and can be collected on an outpatient basis.

Stem cells originating from solid tissue can potentially be applied in tissue repair. Techniques by which new genetic material is introduced into stem cells are being developed, and may lead to the cure of various inherited diseases by somatic gene therapy.

    Acknowledgments

We thank David Kramer, Dana Faratian, and Dona Kocan for scentific help with preparing the manuscript.

    Footnotes

Funding: NIH grants DK49745 and AI41011.

Competing interests: None declared.

    References
Top
Haematopoietic stem cells
Non-haematopoietic stem cells
Conclusions
References

1. Till J, McMulloch E. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Rad Res 1961; 14: 1419-1430.
2. Wu A, Till J, Simminovitch L, McCulloh E. Cytological evidence for a relationship between normal hematopoietic colony-forming cells and cells from the lymphoid system. J Exp Med 1968; 127: 455-467[Abstract].
3. Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. Antigenic analysis of hematopoiesis III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 1984; 133: 157-165[Abstract].
4. Fraser C, Szilvassy S, Eaves C, Humphries R. Proliferation of totipotent hemopoietic stem cells in vitro with retention of long term competitive in vivo reconstituting ability. Proc Natl Acad Sci USA 1992; 89: 1968-1972[Abstract/Free Full Text].
5. Kessinger HH, Armitage JO, Smither DM, Landmark JD, Bierman PJ, Weisenburger DD. High dose chemotherapy and autologous peripheral blood stem cell transplantation for patients with lymphoma. Blood 1989; 74: 1260-1265[Abstract/Free Full Text].
6. Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, et al. Human umbilical cord blood as a potential source of transplantable hemopoietic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: 3828[Abstract/Free Full Text].
7. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci USA 1995; 92: 10302-10306[Abstract/Free Full Text].
8. Byrne JL, Russel NH. Peripheral blood stem cell transplants. J Clin Pathol 1998; 51: 351-355[Medline].
9. Knusten AP, Wall D. Kinetics of T-cell development of umbilical cord blood transplantation in severe T-cell immunodeficiency disorders. J Allergy Clin Immunol 1999; 103: 823-832[Medline].
10. Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, Yoder M, et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992; 89: 4109[Abstract/Free Full Text].
11. Rubinstein P, Adamson JW, Stevens C. The placental/umbilical cord blood program of the New York Blood Center. A progress report. Ann NY Acad Sci 1999; 872: 328-334[Abstract/Free Full Text].
12. Rafii S, Shapiro F, Rimarachin J, Nachan RL, Ferris B, Weksler B, et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 1994; 84: 10-19[Abstract/Free Full Text].
13. Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci USA 1990; 87: 3584-3588[Abstract/Free Full Text].
14. Petzer AL, Zandstra PW, Piret JM, Eaves CJ. Differential cytokine effects on primitive (CD34+ CD38-) human hematopoietic cells: novel response to flt-3-ligand and thrombopoietin. J Exp Med 1996; 183: 2551-2558[Abstract/Free Full Text].
15. Caux C, Massacrier C, Vanbervliet B, Dubois B, De Saint-Vis B, Dezotter-Dambuyant C, et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNFa. J Exp Med 1996; 184: 695-706[Abstract/Free Full Text].
16. Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy. Ann Rev Med 1999; 50: 507-529[Medline].
17. Tjoa BA, Simmons SJ, Elgamal A, Rogers M, Ragde H, Kenny GM, et al. Follow-up evaluation of a phase II prostate cancer vaccine trial. Prostate 1999; 40: 125-129[Medline].
18. Starzl TE, Demetris AJ, Murase N, Ildstad S, Ricordi C, Trucco M. Cell migration, chimerism and graft acceptance. Lancet 1992; 339: 1579-1582[Medline].
19. Thomson AW, Lu L, Murase N, Demetris AJ, Rao AS, Starzl TE. Microchimerism, dendritic cell progenitors and transplantation tolerance. Stem Cells 1995; 13: 622-639[Abstract].
20. Thomson AW, Lu L. Are dendritic cells the key to liver transplantation tolerance? Immunol Today 1999; 20: 27-32[Medline].
21. Lu L, Lee WC, Takayama T, Qian S, Gambotto A, Robbins PD. Genetic engineering of dendritic cells to express immunosuppressive molecules. J Leukocyte Biol 1999; 66: 293-296[Abstract].
22. Cowan KH, Moscow JA, Huang H, Zujewski JA, O'Shaughnessy J, Sorretino B, et al. Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients. Clin Cancer Res 1999; 5: 1619-1628[Abstract/Free Full Text].
23. Rafferty JA, Hickson I, Chinnasamy N, Lashford LS, Margison GP, Dexter TM, et al. Chemoprotection of normal tissues by transfer of drug resistance genes. Cancer Metastasis Rev 1996; 15: 365-383[Medline].
24. Capel B, Hawley RG, Mintz B. Long and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers. Blood 1990; 75: 2267-2270[Abstract/Free Full Text].
25. Bachier CR, Giles RE, Ellerson D, Hanania EG, Garcia-Sanchez F, Andreeff M, et al. Hematopoietic retroviral gene marking in patients with follicular non-Hodgkin's lymphoma. Leukemia Lymphoma 1999; 32: 279-288.
26. Heslop HE, Brenner MK, Krance RA, Bowman L, Cunningham JM, Richardson S, et al. Use of double marking with retroviral vectors to determine rate of reconstitution of untreated and cytokine expanded CD34+ selected marrow cells in patients undergoing autologous bone marrow transplantation. Hum Gene Ther 1996; 7: 655-657[Medline].
27. Podsakoff G, Wong KK, Chatterjee S. Efficient gene transfer into non-dividing cells by adeno-associated virus-based vectors. J Virol 1994; 68: 5656-5666[Abstract/Free Full Text].
28. Flake A, Zanjani ED. In utero hematopoietic stem cell transplantation: a status report. JAMA 1997; 278: 932-937[Abstract].
29. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147[Abstract/Free Full Text].
30. Bruder SP, Ricalton NS, Boynton RE, Connolly TJ, Jaiswal N, Zaia J, et al. Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Min Res 1998; 13: 655-663[Medline].
31. Awad HA, Butler DL, Boivin GP, Smith FN, Malaviya P, Huibregtse B, et al. Autologous mesenchymal stem cell-mediated repair of tendon. Tissue Eng 1999; 5: 267-277[Medline].
32. Magnuson T, Epstein CJ, Silver LM, Martin GR. Pluripotent embryonic stem cell lines can be derived from tw5/tw5 blastocysts. Nature 1982; 298: 750-753[Medline].
33. Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Devel Biol 1998; 38: 133-165[Medline].
34. Martin GR. Isolation of pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 1981; 78: 7634-7638[Abstract/Free Full Text].
35. Solter D, Gearhart J. Putting stem cells to work. Science 1999; 283: 1468-1470[Free Full Text].
36. Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 1993; 90: 8424-8428[Abstract/Free Full Text].
37. Fisher LJ. Neural precursor cells: applications for the study and repair of the central nervous system. Neurobiol Dis 1997; 4: 1-22[Medline].
38. Shihabuddin LS, Ray J, Gage F. Stem cell technology for basic science and clinical applications. Arch Neurol 1999; 56: 29-32[Free Full Text].
39. Bjorklund A, Svendsen C. Stem cells: breaking the brain-blood barrier. Nature 1999; 397: 569-567[Medline].
40. Dunnett SB, Bjorklund A. Prospects for new restorative and neuroprotective treatments in Parkinson's disease. Nature 1999; 399: 32-39.
41. Philpott LM, Kopyov OV, Lee AJ, Jacques S, Duma CM, Caine S, et al. Neuropsychological functioning following fetal striatal transplantation in Huntington's chorea: three case presentations. Cell Transplant 1997; 6: 203-212[Medline].
42. Frisen J, Johansson CB, Lothian C, Lendahl U. Central nervous system stem cells in the embryo and adult. Cellular Molecular Life Sci 1998; 54: 933-945.
43. Foster CS, Ke Y. Stem cells in prostatic epithelia. Int J Exp Path 1997; 78: 311[Medline].
44. Bonkhoff H, Remberger K. Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: a stem cell model. Prostate 1996; 28: 98[Medline].
45. Alison MR, Golding MHC, Sarraf CE. Pluripotential liver stem cells: facultative stem cells located in the biliary tree. Cell Proliferation 1996; 29: 373-402[Medline].
46. Ruck P, Xiao JC, Kaiserling E. Small epithelial cells and the histogenesis of hepatoblastoma: electron microscopic, immunoelectron microscopic and immunohistochemical findings. Am J Pathol 1996; 148: 321-329[Abstract].
47. Hsia CC, Evarts RP, Nakatsukasa H, Marsden ER, Thorgeirsson SS. Occurrence of oval-type cells in hepatitis B virus-associated human hepatocarcinogenesis. Hepatology 1992; 16: 1327-1333[Medline].
48. Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, Rama P, et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 1999; 145: 769-782[Abstract/Free Full Text].
49. Holland EJ, Schwartz GS. Epithelial stem-cell transplantation for severe ocular-surface disease. N Engl J Med 1999; 340: 1752-1753[Free Full Text].
50. Akpek EK, Foster CS. Limbal stem-cell transplantation. Int Ophthalmol Clinics 1999; 39: 71-82.

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