Author: Karina Palomares

Last week, the International Society for Stem Cell Research (ISSCR) held its 11^th Annual Meeting in Boston, MA.  Over 3,000 stem cell researchers from around the world gathered to hear the leading experts share their research and perspectives in this fast-paced field.  One interesting topic was recent advances in cell-replacement therapy for treatment of type 1 diabetes.

Diabetes mellitus is a metabolic disease that results from a failure in glucose regulation, often leading to severe hyperglycemia and tissue/organ damage.  Pancreatic β-cells respond to high blood glucose levels by secreting insulin, which acts on other tissues to promote glucose uptake from the blood.  Type 1 diabetes (T1D) results from autoimmune destruction of insulin-producing β-cells of the pancreas.  The lack of insulin leads to increased blood and urine glucose.  T1D is often fatal unless treated with exogenous administration of insulin daily and regular blood glucose monitoring for the patient’s entire life.  However, this treatment does not match the effect of having endogenous β-cells.  Thus, scientists in the field of regenerative medicine have focused on strategies for generation of β-cells for cell-replacement therapy.

Douglas Melton presented his lab’s current progress on generation of functional β-cells from human embryonic stem cells (hESCs).  Since 2006, D’Amour et al. demonstrated that they had developed a robust differentiation protocol to produce hESC-derived pancreatic endocrine cells capable of synthesizing insulin ¹.  However, the derived cells failed to secrete insulin appropriately in response to the addition of glucose, a required function of true β-cells.  Thus, one focus of Melton’s group has been to identify the signals to generate functional β-cells.  Pancreatic islets are complex structures consisting of multiple cell types, including the insulin-producing β-cells as well as endothelial cells in the surrounding blood vessels.  Thus, endothelial signals are known to promote pancreatic development ².  Melton’s group found that co-culture of β-cells with endothelial cells promotes functional maturation of the hESC-derived β-cells.  In addition, they recently identified another hormone, betatrophin, which is secreted by the liver and functions in promoting β-cell replication ³.  Thus, increasing the levels of this hormone may generate more β-cells.  Although various groups have demonstrated that large amounts of glucose-responsive, insulin-secreting β-cells can be generated in vitro, one concern that remains to be addressed is how the cells will be protected from an autoimmune attack once delivered to the patient.  One potential strategy involves encapsulation of the β-cells into an immunoprotective device prior to delivery.

Other mature cells have also been proposed as a source of new β-cells.  Sarah Ferber presented her lab’s work on inducing liver cells to transdifferentiate into β-cells for autologous cell-replacement therapy.  Transdifferentiation is the process by which one type of adult cell is directly converted into another type of cell.  They used the transcription factor, pancreatic and duodenal homeobox gene 1 (PDX-1), and soluble factors to induce the developmental shift of adult human liver cells into functional insulin-producing cells ⁴.  Not only did the transdifferentiated liver cells produce insulin, but they also released in a glucose-regulated manner.  When transplanted into diabetic, immunodeficient mice, the cells ameliorated hyperglycemia over a 60-day period.  Thus, PDX-1-induced transdifferentiated liver cells offer the potential to replace β-cells’ function in vivo.  Furthermore, transplantation of autologous β-cells would circumvent a host versus graft immune response, as well as allow the patient to be the donor of his or her own insulin-producing cells.

In summary, Melton’s and Ferber’s presentations demonstrated the tremendous progress that has been made in the last decade to generate functional β-cells for use in treatment of T1D.

References

1          D’Amour, K. A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392-1401, doi:10.1038/nbt1259 (2006).

2          Nikolova, G. et al. The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation. Dev Cell 10, 397-405, doi:10.1016/j.devcel.2006.01.015 (2006).

3          Yi, P., Park, J. S. & Melton, D. A. Betatrophin: A Hormone that Controls Pancreatic beta Cell Proliferation. Cell 153, 747-758, doi:10.1016/j.cell.2013.04.008 (2013).

4          Sapir, T. et al. Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells. Proc Natl Acad Sci U S A 102, 7964-7969, doi:10.1073/pnas.0405277102 (2005).

HSC gene therapy is an emerging therapeutic option for several disorders of the blood and immune system.  Ex vivo cell therapies are based on the ability to isolate CD34+ cells from a patient or a normal donor, expansion ex vivo with genetic modification, and systemic administration into the patient following myeloablative treatment.  An efficient method for gene transfer into HSCs is required for successful gene therapy.  Lentiviral vectors (LVs) have emerged as a robust and versatile tool for ex vivo and in vivo gene delivery into multiple cell types, including HSCs.  LVs can either be pseudotyped with viral envelope glycoproteins that confer a broad tropism, such as the vesicular stomatitis virus G (VSV-G) protein, or those that confer a specific HSC tropism, including gibbon ape leukemia virus (GALVTR), feline endogenous retrovirus RD114 (RD114TR), or amphotropic murine leukemia virus (MLV-A) proteins.  However, viral envelopes vary in transduction efficiency.  Thus, transduction protocols often involve the addition of factors to enhance viral entry, including cationic polymers (polybrene) ¹ or fibronectin fragments (Retronectin) ².

Recently, in Molecular Therapy-Nucleic Acids, Fenard et al identified another viral entry enhancer, Vectofusin-1 ³.  Vectofusin-1 is a synthetic, histidine-rich cationic amphipathic peptide derived from the LAH4 peptide family.  LAH4 peptides and their derivatives are known to be efficient DNA transfection agents ⁴.  In this study, the authors examined whether Vectofusin-1 would also enhance gene transfer of LVs into CD34+ cells derived from human umbilical cord blood.  Indeed, Vectofusin-1 significantly increased the transduction efficiency of LVs pseudotyped with various envelopes (VSV-G, GALVTR, RD114TR, MLV), with transduction levels ranging from 50-80% compared to undetectable transduction levels in its absence.  In addition, the increased transduction efficiency was not cytotoxic.  Addition of Vectofusin-1 during transduction of CD34+ cells did not negatively affect subsequent myeloerythroid differentiation in colony-forming cell (CFC) assays in vitro, or hematopoietic reconstitution in immunodeficient BALB-Rag/γC mice in vivo.  The mechanism for the increased transduction efficiency was attributed to insertion of the peptide in the viral and cellular membranes, resulting in an enhancement in both adhesion and fusion of the viral particles with the cell’s plasma membrane.

In short, the authors demonstrated that Vectofusin-1 is a promising LV entry enhancer that can be potentially used in ex vivo transduction of HSCs for subsequent use in clinical applications.  Addition of Vectofusin-1 to the transduction medium had similar effects as the commonly used Retronectin, although the latter is used to coat plates, suggesting a different mechanism of action.  Future experiments will determine whether Vectofusin-1 and Retronectin can be used together to synergistically enhance HSC transduction.

References:

1          Davis, H. E., Morgan, J. R. & Yarmush, M. L. Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97, 159-172 (2002).

2          Pollok, K. E. & Williams, D. A. Facilitation of retrovirus-mediated gene transfer into hematopoietic stem and progenitor cells and peripheral blood T-lymphocytes utilizing recombinant fibronectin fragments. Curr Opin Mol Ther 1, 595-604 (1999).

3          Fenard, D. et al. Vectofusin-1, a new viral entry enhancer, strongly promotes lentiviral transduction of human hematopoietic stem cells. Mol Ther Nucleic Acids 2, e90, doi:10.1038/mtna.2013.17 (2013).

4          Kichler, A., Leborgne, C., Marz, J., Danos, O. & Bechinger, B. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc Natl Acad Sci U S A 100, 1564-1568, doi:10.1073/pnas.0337677100 (2003).

HSCs must continuously self-renew to replenish the pool of mature blood cells throughout the life an adult.  One requirement for extensive self-renewal is high telomerase activity to prevent telomere shortening.  HSCs isolated from adult bone marrow have shorter telomeres than cells from fetal liver or umbilical cord blood ¹, suggesting that proliferative potential may decrease with age.  Also, HSC aging is associated with decreased lymphoid potential, as well as an up-regulation of genes involved in leukemic transformation ².  Consequently, “aging” HSCs may have functional defects that might be detrimental for therapeutic strategies involving genetic manipulation and transplantation of HSCs for the treatment of various blood disorders.

Recently, in Blood, Wahlestedt et al examined whether characteristics of aging HSCs are reversible ⁵.  First, they derived iPSCs from young and aged murine HSCs.  To examine their differentiation potential, they injected the derived iPSCs into murine blastocysts and analyzed the engraftment of the donor cells in the developing chimeric embryos.  Overall, iPSCs derived from aged HSCs demonstrated similar differentiation potential compared to that of younger HSCs.  The engraftment of bone marrow mononuclear cells from primary chimeric mice in a competitive transfer experiment was comparable to that of young HSCs.  Aged HSCs, on the other hand, demonstrated a significant reduction in repopulation capacity.  Interestingly, aged iPSC-derived HSCs also generated naïve T cells at similar levels as young HSCs.

Next, the authors examined telomere length following re-differentiation of the young and aged iPSCs.  Telomeres in aged HSCs were ~11% shorter compared to young HSCs.  However, telomeres of the HSCs derived from the aged iPSCs demonstrated a 2-fold elongation compared to blastocyst control HSCs.  This 2-fold elongation was maintained even after transplantation.  Overall, these results indicate that iPSC induction from HSCs results in elongation of telomeres.

In short, Wahlestedt et al demonstrated that reprogramming does indeed reverse some of the functional defects associated with chronologically aged HSCs, including decreased differentiation potential and shortened telomeres.  However, the study did not address whether the iPSCs derived from aged HSCs had an increased DNA mutation frequency, since HSC aging is also associated with a higher mutation rate.  It would also be interesting to determine whether the above phenomena are also observed in reprogramming of aged human HSCs.  If iPSC induction does indeed result in an “epigenetic reset,” then HSCs derived from iPSCs may have unique characteristics favorable for use in clinical settings.

References

1          Vaziri, H. et al. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci U S A 91, 9857-9860 (1994).

2          Rossi, D. J. et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 102, 9194-9199, doi:10.1073/pnas.0503280102 (2005).

3          Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55-70, doi:10.1016/j.stem.2007.05.014 (2007).

4          Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141-154, doi:10.1016/j.stem.2008.12.010 (2009).

5          Wahlestedt, M. et al. An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state. Blood, doi:10.1182/blood-2012-11-469080 (2013).

The initial finding that pluripotency could be induced in human somatic cells revolutionized the field of regenerative medicine, since patient-specific stem cells can now be generated to further examine the causes and mechanisms of various human diseases.  Since the discovery of human iPSCs in 2007 ¹, various studies have focused on improving the reprogramming methods in order to increase the induction efficiency, as well as to further simplify the protocol.  iPSCs are commonly generated from dermal fibroblasts.  However, skin biopsies are required to isolate fibroblast cells, highlighting the necessity to identify an alternative source of cells for reprogramming that would involve less invasive surgical procedures for isolation.

Cord blood cells and peripheral blood mononuclear cells (PMNCs) are attractive sources for the generation of iPSCs due to the low invasiveness of their collection, as well as the abundance of blood banks for potential donors.  iPSCs were first derived from human peripheral blood in 2009 ².  CD34+ cells were mobilized from peripheral blood and subsequently transduced with retroviruses delivering OCT4, SOX2, KLF4, and MYC vectors.   Although the reprogramming was successful, use of retroviral vectors requires genomic integration of transgenes that may increase the risk of tumor formation during clinical applications.  Thus, recent studies have focused on generation of “integration-free” iPSCs.  Yet, development of integration-free methods often means compromising the reprogramming efficiency.  iPSC induction in CD34+ cells using non-integrating episomal plasmids resulted in ~0.03% reprogramming efficiency ³.

Recently, in Stem Cells, Yamanaka’s group reported a protocol that increased the efficiency of iPSC induction from CD34+ cord blood and peripheral blood ⁴.  They previously identified an efficient combination of episomal plasmids for reprogramming of adult fibroblasts, termed the “Y4” combination, consisting of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and an shRNA for TP53 ⁵.  Transfection of CD34+ cells from human cord blood with the Y4 combination resulted in up to 0.1% reprogramming efficiency across two donors.  iPSC induction efficiency of PMNCs isolated from peripheral blood with the Y4 mixture, on the other hand, was inconsistent across donors.  To further increase the reproducibility of iPSC induction from multiple donors, the authors added a vector encoding EBNA1, which is required for episomal plasmid replication and should thereby increase expression of the episomal plasmids.  Addition of the EBNA1 vector to the Y4 mixture resulted in 0.1% reprogramming efficiency in PMNCs across seven donors.  Both, the CD34⁺– and PMNC-derived iPSCs were molecularly and functionally identical to hESCs.

In summary, Okita et al identified a new protocol allowing efficient generation of integration-free iPSCs from blood.  Previous studies reported ~0.02%- 0.03% induction efficiency from peripheral blood ² and isolated CD34+ cells ³.  Here, the authors reported a reprogramming efficiency of ~0.06%, with a maximum of 0.1%.  In addition, the new protocol could induce iPSCsfrom frozen PMNCs as efficiently as from freshly isolated cells.  Thus, with the increasing number of potential donors available at cord blood banks, iPSCs can be now efficiently generated for use in autologous or allogeneic stem cell therapy.

References

1          Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872, doi:10.1016/j.cell.2007.11.019 (2007).

2          Loh, Y. H. et al. Generation of induced pluripotent stem cells from human blood. Blood 113, 5476-5479, doi:10.1182/blood-2009-02-204800 (2009).

3          Mack, A. A., Kroboth, S., Rajesh, D. & Wang, W. B. Generation of induced pluripotent stem cells from CD34+ cells across blood drawn from multiple donors with non-integrating episomal vectors. PLoS One 6, e27956, doi:10.1371/journal.pone.0027956 (2011).

4          Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458-466, doi:10.1002/stem.1293 (2013).

5          Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat Methods 8, 409-412, doi:10.1038/nmeth.1591 (2011).

Mature blood cells are relatively short-lived, and require replenishment from multipotent HSCs. Thus, HSCs must self-renew to generate an adequate pool of HSCs, as well as differentiate to give rise to more mature blood cells.  A balance between self-renewal and differentiation ensures that the hematopoietic system can be functionally sustained throughout the lifetime of an adult body.  However, as HSCs age, they accumulate DNA damage, often compromising their functionality.  DNA damage can be further propagated both to daughter stem cells and downstream lineages, and may increase the risk of developing blood disorders ¹.

Depending on the nature of the damage, cells use two major response pathways to combat cellular stress.  If the damage is excessive and functionality is compromised, cells usually undergo apoptosis for self-elimination.  In contrast, autophagy allows cells a window of survival.  Autophagy is a process of self-degradation in which organelles or portions of the cytoplasm are sequestered within double-membrane vesicles, known as autophagosomes, and then delivered to lysosomes for degradation ².  The resulting breakdown products are released through permeases and recycled in the cytosol.  Thus, autophagy can be used to generate high-energy compounds during conditions of metabolic stress.

Recently, in Nature, Warr et al found that metabolic stress and old age induce autophagy in HSCs ³.  The authors isolated HSCs and myeloid progenitors from the bone marrow of transgenic mice systemically expressing GFP fused to LC3, an autophagosome marker ⁴.  They used cytokine withdrawal to induce metabolic stress and measured autophagy induction by examining the formation and turnover of LC3-GFP.  Myeloid progenitors expressed LC3-GFP in the presence and absence of cytokines.  In contrast, HSCs did not express LC3-GFP in the presence of cytokines, but demonstrated autophagosome formation following cytokine withdrawal.  Furthermore, when the mice were starved in vivo, autophagy flux increased in HSCs.

The authors speculated that autophagy “protects” HSCs from starvation-induced apoptosis, and indeed, hematopoietic-specific deletion of an essential autophagy machinery component, ATG12, resulted in a significant increase in caspase activation in starved HSCs in vivo.  FOXO3A was identified as the specific transcriptional regulator that maintains pro-autophagy gene expression, and was expressed higher in HSCs compared to progenitors.  Interestingly, HSCs isolated from old mice retained their autophagic potential, and was found to be required for their survival.

In summary, Warr et al demonstrated that long-lived HSCs mount a protective survival autophagy response to combat metabolic stress, whereas short-lived progenitors do not.  Previous studies suggested that impaired autophagy might contribute to the aging phenotype ⁵.  However, this study directly showed that the pro-autophagy gene expression program is still intact in old HSCs and is essential for continued survival of these cells.  Future studies will address whether autophagy increases the incidence of age-related blood disorders since it protects damaged, old HSCs from elimination by apoptosis.

References 

1          Rossi, D. J., Jamieson, C. H. & Weissman, I. L. Stems cells and the pathways to aging and cancer. Cell 132, 681-696, doi:10.1016/j.cell.2008.01.036 (2008).

2          He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43, 67-93, doi:10.1146/annurev-genet-102808-114910 (2009).

3          Warr, M. R. et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells. Nature 494, 323-327, doi:10.1038/nature11895 (2013).

4          Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15, 1101-1111, doi:10.1091/mbc.E03-09-0704 (2004).

5          Rubinsztein, D. C., Marino, G. & Kroemer, G. Autophagy and aging. Cell 146, 682-695, doi:10.1016/j.cell.2011.07.030 (2011).