Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04

    • This finding is consistent with our observations

    2018-10-22

    This finding is consistent with our observations in human fetal RG cells (Mo and Zecevic, 2008), indicating that in both cell populations Pax6 affects proliferation as well as neuronal fate. Numerous studies have reported the successful transplantation of hESC into rodent brains (Kelly et al., 2004; Roy et al., 2006; Yang et al., 2008). However, far less is known about how hESC behave when transplanted into the human histamine receptors (Lindvall and Kokaia, 2006). This knowledge, however, is very important for the development of future therapies. In the present study, we created a co-culture system in an attempt to mimic the cellular interactions that may occur upon transplantation of hESC-RG cells in the human fetal forebrain. Our experiments demonstrated that the microenvironment of the human fetal forebrain (17–22gw) promotes neurogenesis in hESC-RG cells when compared to the commonly-used substrate, poly-l-ornithine-laminin (control). In the presence of the same differentiation medium, the number of neurons derived from hESC-RG cells was nine fold higher in co-cultures compared to the control cultures. At the same time the number of derived astroglia decreased more than six fold compared to control cultures. This is in line with our previous report that RG isolated from mid-gestational human fetal forebrain generate more neurons than glia (Mo et al., 2007). Neuronal fate of co-cultured hESC-RG was determined by general neuronal markers, such as βIII-tubulin and MAP2. Notably, these cells were functionally evaluated by patch-clamp recordings. Three lines of evidence strongly indicate that cells derived from hESC-RG are committed to the neuronal fate. First, these cells exhibited typical neuronal morphologies (Fig. 6A3). Second, the peak sodium current exceeded 200pA (Fig. 6B2) (Moore et al., 2009). Third, upon depolarization, the GFP+ cells produced regenerative spikes (abortive AP, full-size AP, and repetitive APs, Fig. 6C2). With each day of in vitro cultivation, the two parameters (sodium current and AP firing pattern) clearly shifted towards values characteristic of more mature neurons (Figs. 6B1C1). Mature forebrain neurons can be separated between GABA-ergic inhibitory and non-GABA-ergic neuron types solely based on their characteristic action potential firing pattern (Connors and Gutnick, 1990). In immature human neurons (4weeks in vitro), such separation is considerably less reliable because the sodium and potassium channels are not fully expressed in the plasma membrane (Moore et al., 2009; Belinsky et al., 2011). As a consequence of low channel density the AP amplitudes do not overshoot in repetitive firing (Fig. 6C2, Repetitive-1 and Repetitive-2). We think that voltage waveforms termed “Single AP” and “Repetitive-2” (Fig. 6C2) pertain to young glutamatergic neurons, while a delayed-onset high-frequency AP-firing pattern (Repetitive-1) is more indicative of immature fast-spiking GABA-ergic interneurons (Gupta et al., 2000). Significantly, various neuronal subtypes were generated from hESC-RG in our co-culture system. More than half of the generated neurons by their molecular markers were projection, pyramidal neurons (SMI-32+ and Tbr1+), but other subtypes, such as interneurons (GABA+ and calretinin+) and dopaminergic neurons (TH+) were also derived from hESC-RG under these conditions. Based on the expression of various patterning markers, such as rostral and dorsal forebrain markers (Foxg1, Otx2, Pax6 and Tbr2), but also caudal and ventral markers (Pax7 and HoxC8) (Figs. 1J, K), the hESC-RG cell population is likely a mixed population of both dorsal and ventral progenitors. This is further confirmed by their differentiation into various neuronal cell types of either dorsal (SMI-32, Tbr1) or ventral (TH, GABA, calretinin) origin (Fig. 5). In our hESC-RG cultures, the observed increase in neurogenesis was, at least partially, due to secreted factors, because the neurogenic effect was produced with the medium conditioned with the human fetal forebrain alone. The major difference between media collected from control cultures (grown on poly-l-ornithine-laminin) and CM from co-cultures, is the presence of astrocytes and neurons in co-cultures; both cell types can release factors, including growth factors and cytokines, that may have a role in the observed increase in the neurogenic fate of GFP hESC-RG.