Brain Aging and hESC-derived Neural Stem Cell Transplantation

Funding Type: 
SEED Grant
Grant Number: 
ICOC Funds Committed: 
Disease Focus: 
Blood Cancer
Stem Cell Use: 
Cancer Stem Cell
Embryonic Stem Cell
Cell Line Generation: 
Cancer Stem Cell
Public Abstract: 
Aging is an important risk factor for human diseases. In addition to cognitive decline in the elderly, brain aging also increases the risk for neurological diseases like Alzheimer’s disease (AD), Parkinson's disease (PD), and stroke, which are major causes of disability and mortality in the elderly. Current therapeutic approaches typically fail to treat the underlying cause of these disorders. The potential capacity of human neural stem cells to replace cells and tissues damaged due to aging and age-realted diseases is, therefore, of great importance. Activation or transplantation of these cells has already shown promise in animal models of these disorders. However, little is known about how aging affects host receptivity to hESC-derived cell transplants, or about the proliferation, survival, differentiation, migration and functionality of the donor cells. This is partly because in vivo studies of aged-related neurological diseases have relied almost universally on experimental models using young adult animals.We hypothesize that the ability of hESC-derived transplants to proliferate, survive, differentiate, migrate and function may be compromised in the aged brain, requiring that new strategies be devised to overcome this deficiency. The experiments we propose are designed to increase our understanding of how the age of the recipient alters the ability of transplanted neural stem cells to differentiate into mature, functional neurons and integrate into local neuronal circuits. Our first Specific Aim will examine these issues using immunocytochemistry and electrophysiological methods, after transplanting neural stem cells into young adult, middle aged and aged rats. In addition, because brain aging produces progressive changes in learning and memory, a critical area to examine is whether neural stem cell therapy can produce functional improvement in learning and memory deficits in aged rats. Our second Specific Aim will address this question by using a battery of behavioral tests to assess the ability of transplanted neural stem cells to reverse age-related losses of cognitive function. The use of neural stem cells to repair age-related neurological diseases will require an increased understanding of stem cell biology, the environment of the aged tissue, and the interaction between the two, which this proposed work will focus on. If the aims of the application are achieved, significant advances will be made in establishing a new paradigm of brain aging and the biological behaviors of transplanted neural stem cells, and substantial progress will be made in developing basic science knowledge that can be translated fairly rapidly into clinical treatment of aged-related neurological diseases.
Statement of Benefit to California: 
Aging is destined to become a serious public health problem for California over the next several decades. California’s elderly population is expected to grow more than twice as fast as the total population from 1990 to 2020, and will reach 12.5 million by 2040. One in five Californians will be 60 years of age or older beginning in 2010. Consequently, age-related diseases, including neurological diseases, will dramatically increased in parallel, presenting enormous social and economic challenges. Determining whether transplanted human neural stem cells can differentiate into functional neurons in the aging brain, and thereby slow or prevent cognitive decline in the aged, could have great social and economic impact in our state.
Progress Report: 
  • SEED Grant Research Summary
  • Compelling studies suggest that cancer stem cells (CSC) arise from primitive self-renewing progenitor cells. Although many cancer therapies target rapidly dividing cells, CSC may be quiescent i.e. asleep resulting in therapeutic resistance. Recently, we demonstrated that CSC drive progression of chronic phase (CP) chronic myeloid leukemia (CML), a subject of many landmark cancer research discoveries, to a therapeutically recalcitrant myeloid blast crisis (BC) phase. CML CSC share cell surface markers with granulocyte-macrophage progenitors (GMP) and have amplified expression of the CML fusion gene, BCR-ABL. In addition, they aberrantly gain self-renewal capacity, in part, as a result Wnt/β-catenin activation. Because human embryonic stem cells (hESC) have robust regenerative capacity and can provide a potentially limitless source of tissue specific progenitor cells in vitro, they represent an ideal model system for generating and characterizing human CSC. The main goals of this research were to generate CSC from hESC to provide an experimentally amenable platform to expedite the development of sensitive diagnostics that predict progression and combined modality anti-CSC therapy.
  • To this end, we tested whether BCR-ABL expression in hESC is sufficient to induce changes characteristic of CML stem cells. Unlike mouse ESC, introduction of a novel lentiviral BCR-ABL vector into hESC did not drive myeloid differentiation nor did it induce stromal independence in vitro underscoring key differences between mouse and human hESC and the importance of in vivo models. Notably, Hues16 cells had a higher propensity to differentiate into CD34+ cells than other hESC lines particularly in AGM co-cultures and thus, were used in subsequent in vivo experiments. Moreover, this SEED grant funded Yosuke Minami in Professor Jean Wang’s lab to create a unique CML blast crisis mouse model typified by GMP expansion and resistance to a BCR-ABL inhibitor, imatinib (Minami et al, PNAS 2008;105:17967-72). In addition, a bioluminescent humanized model of blast crisis CML was created based on transplantation of GMP from patient blood into immune deficient mice (RAG2-/-gc-/-). Cells were tagged with firefly luciferase that emits a bioluminescent signal so that leukemic transplantation efficiency could be tracked in vivo (IVIS). As few as 1,000 human blast crisis CML GMP could transplant leukemia in immune deficient mice thereby providing an important model for studying the molecular events that contribute to leukemic transformation (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the second aim, we hypothesized that BCR-ABL is sufficient for generating CML from self-renewing stem cells. In these studies, Hues16 cells differentiated into CD34+ cells were lentivirally transduced with BCR-ABL leading to sustained BCR-ABL engraftment in 50% of transplanted mice. Chronic phase CD34+ cells derived from CML blood were less efficient at sustaining CML engraftment (7%) suggesting that hESC derived CD34+ cells have higher self-renewal potential and are similar to advanced phase CML progenitors.
  • Thirdly, we hypothesized that BCR-ABL was necessary but not sufficient for progression to blast crisis. Introduction of lentiviral activated beta-catenin or shRNA to GSK3beta, together with BCR-ABL did not enhance BCR-ABL engraftment compared with BCR-ABL transduction of hESC alone. These studies suggested that hESC may already have sufficient self-renewal capacity to sustain the malignant CML clone and are molecularly comparable to advanced CML progenitors that behave like CSC. In addition, through extensive cDNA sequencing of human blast crisis CML progenitors, we found that 57% of samples harbored a misspliced form of GSK3beta that promoted tumor production and could serve as a novel prognostic marker in CML clinical trials (Abrahamsson et al, PNAS 2009;106:3925-9).
  • In the final aim, we hypothesized that CML CSC are not eliminated by BCR-ABL inhibitors alone and that combined modality therapy will be required. In collaborative research involving in vitro analysis of imatinib resistant CML progenitors and more recently in a humanized mouse model of blast crisis CML, we found that dasatinib, a potent BCR-ABL inhibitor, is necessary but not sufficient for CSC eradication. Discovery of a GSK3beta deregulation, a negative regulator of both beta-catenin and sonic hedgehog (Shh) pathways (Zhang et al, Nature 2009), led us to disover that Shh combined with BCR-ABL inhibition abrogated CSC driven tumor formation (manuscript in preparation) providing the impetus for an upcoming Pfizer sponsored Shh inhibitor clinical trial for refractory hematologic malignancies.

© 2013 California Institute for Regenerative Medicine